WO2022242931A1 - Verfahren zur aufreinigung von nukleinsäuren, insbesondere in einer mikrofluidischen vorrichtung - Google Patents
Verfahren zur aufreinigung von nukleinsäuren, insbesondere in einer mikrofluidischen vorrichtung Download PDFInfo
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- WO2022242931A1 WO2022242931A1 PCT/EP2022/057426 EP2022057426W WO2022242931A1 WO 2022242931 A1 WO2022242931 A1 WO 2022242931A1 EP 2022057426 W EP2022057426 W EP 2022057426W WO 2022242931 A1 WO2022242931 A1 WO 2022242931A1
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- Prior art keywords
- nucleic acids
- transport medium
- binding
- filter
- washing buffer
- Prior art date
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 63
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 63
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000006163 transport media Substances 0.000 claims abstract description 50
- 239000011534 wash buffer Substances 0.000 claims abstract description 45
- 239000000126 substance Substances 0.000 claims abstract description 33
- 230000003196 chaotropic effect Effects 0.000 claims description 42
- 150000003839 salts Chemical class 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 14
- 150000005846 sugar alcohols Polymers 0.000 claims description 10
- 239000012149 elution buffer Substances 0.000 claims description 9
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000003752 polymerase chain reaction Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 238000011901 isothermal amplification Methods 0.000 claims description 3
- 238000007834 ligase chain reaction Methods 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 description 19
- 239000012148 binding buffer Substances 0.000 description 11
- 239000000523 sample Substances 0.000 description 9
- 239000000872 buffer Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002032 lab-on-a-chip Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 159000000009 barium salts Chemical class 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical class OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- -1 silica nucleic acids Chemical class 0.000 description 1
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 1
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 150000003567 thiocyanates Chemical class 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
-
- 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
- B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
-
- 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
- B01L2200/16—Reagents, handling or storing thereof
-
- 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
Definitions
- transport media for viral and bacterial samples in particular have established themselves as clinical standards, which make it possible to transport patient swabs or samples to a diagnostic laboratory without damaging or changing the nucleic acids in the medium.
- These transport media create a stabilizing environment for the nucleic acids by destroying pathogens, releasing the nucleic acids and inhibiting degrading proteins. They are mainly used when a molecular diagnostic detection of pathogens is to be carried out, but no microbiological multiplication on nutrient media such as agar plates or blood cultures is desired or possible (e.g. for virus detection).
- eNATTM A widespread transport medium for such a purpose is eNATTM from COPAN.
- Another example is Roche's cobas® medium.
- These nucleic acid-stabilizing transport media contain so-called chaotropic salts in high concentrations (e.g. eNATTM: 3.7 M), which disrupt the structure of water, but also of macromolecules dissolved in water, such as proteins or nucleic acids, and can lead to denaturation of the structures.
- Lipid bilayers such as those found in cellular membranes, can also be denatured. This effect leads to the lysis of a wide variety of cells, such as human cells and bacteria, and also to disruption of the protein envelope of viruses.
- the classic sample purification process common in a lab-on-chip system comprises cell lysis, binding of the released nucleic acids to a filter frit, washing and subsequent elution of the purified nucleic acids with subsequent (real-time) polymerase chain reaction (PCR for short) and detection reaction, such as described, for example, in the published application DE 102014211 221 A1, can in principle be carried out with these transport media.
- a so-called lysis or binding buffer is used for cell lysis and binding of the nucleic acids to the filter frit. This buffer is formulated such that the concentration of chaotropic salts in the mixed compound is optimal for binding the nucleic acids to the filter frit.
- this optimum range is approximately 1.6-2.2 moles per liter (Molar or M for short).
- this binding buffer usually contains reagents that promote the precipitation of the nucleic acids. These are often other salts (e.g. table salt) or alcohols (e.g. isopropanol). The setting of these chaotropic conditions is essential for the recovery rate of the nucleic acids.
- wash buffer In a second step, cell and protein residues as well as salts and precipitating agents are removed as completely as possible by using a washing buffer. This is necessary to prevent these substances from inhibiting the detection reaction by a (quantitative real-time) PCR.
- the formulation of the wash buffer must be chosen carefully in order to sufficiently remove interfering substances without rinsing the nucleic acids from the filter and thus losing them for the detection reaction.
- a cascade of solutions of different concentrations with chaotropic salts and precipitating agents is often used sequentially instead of a single wash buffer.
- the nucleic acids are removed from the filter frit with a so-called elution buffer and fed to the detection reaction.
- a freeze-dried PCR reaction mix is rehydrated with the elution buffer and the nucleic acids it contains, which can then be cycled in a classic PCR temperature program. Disclosure of the Invention Advantages of the Invention
- the invention relates to a method for purifying nucleic acids.
- the method can be carried out in particular with a microfluidic device, preferably a so-called lab-on-a-chip, for example with a microfluidic cartridge as described in DE 10 2016 222 075 A1 or DE 10 2016 222 072 A1.
- nucleic acids are released from a sample in a transport medium due to chaotropic substances in the transport medium.
- the transport medium can thus be a medium for storing, preserving and/or transporting biological samples, in particular samples of body fluids containing human or animal cells.
- the transport medium has chaotropic substances, in particular chaotropic salts, for releasing nucleic acids from the cells.
- the transport media mentioned above are eNATTM or cobas®.
- the release can take place in particular by lysis of the cells in the sample, with the cells comprising the nucleic acids.
- the release can include a denaturation of the nucleic acids.
- Nucleic acids can be understood in particular as meaning ribonucleic acids (RNA for short) or deoxyribonucleic acids (DNA for short).
- the chaotropic substances can in particular be chaotropic salts, such as barium salts, guanidinium hydrochloride, thiocyanates such as guanidinium thiocyanate, perchlorates such as sodium perchlorate or else sodium chloride.
- nucleic acids are bound to a filter, with the transport medium being mixed with a first part of a wash buffer to set binding conditions.
- Mixing creates a binding buffer for binding the nucleic acids to the filter.
- the filter can be a filter of the microfluidic device in particular.
- the filter can, for example, on a porous silica membrane.
- a filter can preferably also be understood to mean a filter frit.
- the washing buffer can be a buffer or solution customary for the purification of nucleic acids, but preferably without ethanol and the washing buffer preferably has less than 0.1 mol per liter of chaotropic substances, in particular chaotropic salts.
- Mixing the transport medium with the first part of the washing buffer preferably lowers a concentration of the chaotropic substances for setting the binding conditions, and a binding buffer with a concentration of chaotropic substances between 1.6 and 2.2 mol per liter is preferably created.
- a binding buffer with a concentration of chaotropic substances between 1.6 and 2.2 mol per liter is preferably created.
- mixing the transport medium with the first part of the washing buffer preferably creates a mixture which has chaotropic salts with a concentration of between 1.6 and 2.2 moles per liter.
- the first part of the washing buffer contains highly crosslinked polyalcohols, such as polyethylene glycols, so that the mixture preferably contains these polyalcohols in a proportion of 150 to 200 grams per liter (g/L for short).
- the washing buffer preferably has a proportion of 200 to 400 g/l of highly crosslinked polyalcohols.
- the first part of the washing buffer has such an amount of chaotropic substances, preferably chaotropic salts, and preferably highly crosslinked polyalcohols that mixing the first part of the washing buffer with a specified amount of the specified transport medium creates a mixture which is 1.6 to 2.2 moles per liter of chaotropic substances, where the chaotropic substances are preferably chaotropic salts, and 150 to 200 grams per liter of highly crosslinked polyalcohols, where the highly crosslinked polyalcohols are preferably polyethylene glycol.
- the method according to the invention has the advantage that a binding buffer that would otherwise be required for binding the nucleic acids to the filter can be dispensed with, since this binding buffer is replaced by mixing the transport medium with the first part of the washing buffer.
- a binding buffer for the inventive mixing of the transport medium with the first part of the wash buffer Binding of the nucleic acids created on the filter when carrying out the method.
- the resulting elimination of a separate binding buffer advantageously leads to cost savings both in the procurement and formulation of reagents and buffers and in the design and manufacture of a microfluidic device used for this purpose, since space for receiving and pre-storage and thus material can be saved .
- the purification of the nucleic acids after binding to the filter includes washing the filter with a second part of the washing buffer.
- This has the advantage that substances remaining on the filter or in the filter chamber, for example cell and protein residues and in particular also salts and precipitating agents, are removed and thus cannot adversely affect the further process. In particular, this allows substances to be removed which would adversely affect the implementation of a polymerase chain reaction.
- the first part and the second part of the wash buffer can preferably be two parts of the same wash buffer.
- the nucleic acids are eluted from the filter using an elution buffer.
- the elution buffer can be a buffer for the elution of nucleic acids that is typical in microfluidics, for example buffered low-salt solutions with a neutral to slightly alkaline pH (for example TE buffer 10 mM TRIS/CI pH 8, 1 mM EDTA) or distilled water.
- parts of the nucleic acids are amplified after the binding, in particular after the elution, preferably with the aid of a (quantitative real-time) polymerase chain reaction, an isothermal amplification or a ligase chain reaction.
- a part of the nucleic acids can be understood as meaning a section, also called a sequence, of a nucleic acid, for example a DNA or RNA section.
- a detection of the duplicated parts can provide evidence of these parts and thus the associated organisms, for example with the aid of (fluorescence) spectroscopy.
- the invention also relates to a microfluidic kit or microfluidic system comprising a microfluidic device and a transport medium, the transport medium containing chaotropic substances for the release of nucleic acids from a sample and the device being set up to mix a first part of a washing buffer with the transport medium for setting To mix binding conditions for binding the released nucleic acids to a filter of the device.
- the wash buffer can be stored upstream in the microfluidic device.
- the transport medium can be a medium as explained above, in particular eNATTM or cobas®.
- the microfluidic kit can in particular be understood to mean the microfluidic device together with the transport medium, with the transport medium not yet having to be located in the microfluidic device.
- the transport medium can, for example, be arranged in a vessel, in which case the sample for mixing with the transport medium can also be added to the vessel, for example by inserting a swab containing the sample into the transport medium.
- the transport medium and the first part of the washing buffer are preferably coordinated with one another in terms of their respective amount, their composition and a concentration of the chaotropic substances such that the transport medium for the release of nucleic acids from a sample and that mixing of the transport medium with the first part of the Washing buffer are set up to set binding conditions for binding the released nucleic acids to the filter
- binding conditions there are particular binding conditions when the mixture has a concentration of between 1.6 and 2.2 mol per liter of chaotropic substances, preferably chaotropic salts, and very preferably contains 150 to 200 grams per liter of highly crosslinked polyalcohols, wherein the highly crosslinked polyalcohols are preferably polyethylene glycol.
- the kit in particular the microfluidic device, comprises a second part of the Washing buffer, wherein the second part is designed for washing the filter and the nucleic acids bound to the filter.
- the second part has the same composition as the first part of the wash buffer.
- FIG. 2 shows a flow chart of an exemplary embodiment of the method according to the invention.
- Figures la and lb show an embodiment of the microfluidic kit 200 according to the invention, with which the method 500 according to the invention can be carried out, for example.
- Figure 2 shows a flowchart 500 for the exemplary embodiment of the method 500 according to the invention described below.
- the microfluidic kit 200 includes a microfluidic device 100, for example a microfluidic cartridge 100 as part of a lab-on-a-chip device, as described in DE 102016222 075 A1 or DE 102016222 072 A1.
- a biological sample is introduced into an input chamber 110 of the cartridge 100 in a transport medium 10 via an opening 111 .
- the biological sample can in particular be a body fluid such as blood, sputum, urine or a smear containing human or animal cells.
- the transport medium 10 contains chaotropic substances for releasing the nucleic acids from the cells according to a step 501 of the method 500.
- the transport medium 10 can be eNATTM from the company COPAN or cobas® from the company Roche.
- the transport medium 10 contains, for example, approximately 3.7 moles per liter (molar or M for short) of guanidinium thiocyanate as a chaotropic salt and a total volume of, for example, 300 microliters (m/_ for short).
- the wash buffer 20 is preferably a buffer without chaotropic substances or with only a low concentration of chaotropic substances, preferably with a concentration of less than 0.1 M.
- it is a wash buffer without ethanol, as in EP 2 163 621 A1, and for example comprising polyethylene glycol in a concentration between 100 and 600 g/L as described in DE 10 2014211 221 A1.
- the cartridge 100 also includes a filter 141 which is fluidically connected to the input chamber 110 and the buffer chamber 120 and which can be arranged in a further chamber 140 (filter chamber 140 for short).
- a filter 141 which is fluidically connected to the input chamber 110 and the buffer chamber 120 and which can be arranged in a further chamber 140 (filter chamber 140 for short).
- the released nucleic acids are bound to the filter 141, for which purpose the transport medium 10 is mixed with a first part 21 of the washing buffer 20 to set suitable binding conditions, and the filter 141 mixes this mixture, which now corresponds to a binding buffer 50. is exposed for a period of, for example, 15 to 60 seconds, as shown in Figure lb.
- the first part 21 of the wash buffer 10 provided for in combination with the transport medium 10 is designed or formulated in such a way that, in addition to setting the Binding conditions after mixing with the transport medium 10 can also be used for the subsequent removal of chaotropic substances and other substances that inhibit the subsequent analysis, in particular for carrying out a (quantitative real-time) polymerase chain reaction, in its function as a washing buffer.
- the washing buffer is designed in such a way that it contains few or no chaotropic substances and at the same time highly crosslinked polyalcohols such as polyethylene glycols to support the nucleic acid precipitation (for example in the range of 20 to 40% (w/v)), so that it can the washing buffer 20 can advantageously be used both, after mixing with the transport medium 10 to generate the binding buffer, for binding, and as a normal washing buffer for displacing the chaotropic substances in a washing step.
- highly crosslinked polyalcohols such as polyethylene glycols to support the nucleic acid precipitation
- eNATTM contains about 3.7 M guanidinium thiocyanate as a chaotropic salt as a main component that helps determine the binding properties for binding the nucleic acids to the filter 141 .
- 300 microliters of eNATTM are also used to detect a wide variety of pathogens.
- the final concentration of chaotropic salts for binding the nucleic acids to the filter 141 should be between 1.6 and 2.2M. These suitable binding conditions are achieved by diluting the eNATTM with the first part 21 of the wash buffer 20 if the wash buffer contains no or few chaotropic substances as described above.
- the filter 141 and the nucleic acids bound to it are washed with a second part 22 of the washing buffer 20 in order to remove residues of the binding buffer 50 and other substances from the sample and the transport medium 10, in particular remaining chaotropic substances remove, which would have an adverse effect on the further procedure.
- an elution buffer 40 is used to remove the binding conditions and thus to release the nucleic acids from the filter 141.
- the elution buffer 30 can be placed in a further chamber 130 fluidically connected to the filter chamber 140 (short Elution buffer chamber 130) be upstream.
- the elution buffer can be a typical buffer for dissolving silica nucleic acids, for example distilled or buffered water, optionally with surfactants, such as polysorbate 80 (Tween® 80).
- the purification of the nucleic acids is completed in the fourth step 504 and further processing of the nucleic acids can take place in a preferred fifth step 505, for example amplification of parts of the nucleic acids using a (quantitative real-time) polymerase chain reaction, isothermal amplification or a ligase chain reaction in a further chamber 150 (analysis chamber 150 for short) for detecting the nucleic acids and associated pathogens in the sample.
- a further chamber 150 analysis chamber 150 for short
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Abstract
Description
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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CN202280036069.2A CN117413058A (zh) | 2021-05-17 | 2022-03-22 | 用于纯化核酸,特别是在微流体装置中纯化核酸的方法 |
EP22717555.1A EP4341396A1 (de) | 2021-05-17 | 2022-03-22 | Verfahren zur aufreinigung von nukleinsäuren, insbesondere in einer mikrofluidischen vorrichtung |
US18/562,025 US20240240172A1 (en) | 2021-05-17 | 2022-03-22 | Process for Purifying Nucleic Acids, in particular in a Microfluidic Apparatus |
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Citations (6)
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EP1566437A1 (de) * | 2004-02-20 | 2005-08-24 | Roche Diagnostics GmbH | Adsorption von Nukleinsäuren an eine Festphase |
WO2007106579A2 (en) * | 2006-03-15 | 2007-09-20 | Micronics, Inc. | Integrated nucleic acid assays |
EP2163621A1 (de) | 2008-09-03 | 2010-03-17 | Qiagen GmbH | Verfahren zum Isolieren und Reinigen von Nukleinsäuren |
DE102014211221A1 (de) | 2014-06-12 | 2015-12-17 | Robert Bosch Gmbh | Verfahren zur Anreicherung und/oder Aufreinigung von Nukleinsäuren |
DE102016222072A1 (de) | 2016-11-10 | 2018-05-17 | Robert Bosch Gmbh | Vorrichtung und Verfahren zur geneigten Prozessierung von mikrofluidischen Kartuschen |
DE102016222075A1 (de) | 2016-11-10 | 2018-05-17 | Robert Bosch Gmbh | Prozessiersystem und Verfahren zur Prozessierung einer mikrofluidischen Kartusche mit einer Prozessiereinheit |
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2021
- 2021-05-17 DE DE102021204952.4A patent/DE102021204952A1/de active Pending
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2022
- 2022-03-22 EP EP22717555.1A patent/EP4341396A1/de active Pending
- 2022-03-22 WO PCT/EP2022/057426 patent/WO2022242931A1/de active Application Filing
- 2022-03-22 US US18/562,025 patent/US20240240172A1/en active Pending
- 2022-03-22 CN CN202280036069.2A patent/CN117413058A/zh active Pending
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EP1566437A1 (de) * | 2004-02-20 | 2005-08-24 | Roche Diagnostics GmbH | Adsorption von Nukleinsäuren an eine Festphase |
WO2007106579A2 (en) * | 2006-03-15 | 2007-09-20 | Micronics, Inc. | Integrated nucleic acid assays |
EP2163621A1 (de) | 2008-09-03 | 2010-03-17 | Qiagen GmbH | Verfahren zum Isolieren und Reinigen von Nukleinsäuren |
DE102014211221A1 (de) | 2014-06-12 | 2015-12-17 | Robert Bosch Gmbh | Verfahren zur Anreicherung und/oder Aufreinigung von Nukleinsäuren |
DE102016222072A1 (de) | 2016-11-10 | 2018-05-17 | Robert Bosch Gmbh | Vorrichtung und Verfahren zur geneigten Prozessierung von mikrofluidischen Kartuschen |
DE102016222075A1 (de) | 2016-11-10 | 2018-05-17 | Robert Bosch Gmbh | Prozessiersystem und Verfahren zur Prozessierung einer mikrofluidischen Kartusche mit einer Prozessiereinheit |
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EP4341396A1 (de) | 2024-03-27 |
US20240240172A1 (en) | 2024-07-18 |
CN117413058A (zh) | 2024-01-16 |
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