Method and device for purifying nucleic acids, in particular for nucleic acid amplification

Abstract

The invention relates to a method (500) for purifying nucleic acids from cells in a sample (10) using a microfluidic device (100) and a microfluidic device (100) configured therefor.

Description

State of the art

[0001] To detect pathogens, nucleic acids can be isolated from a sample containing biological cells, a process known as nucleic acid extraction. These acids can then be selectively identified as belonging to a specific pathogen, particularly through nucleic acid amplification such as PCR. The use of such methods at the point of care and as immediate near-patient diagnostics can be implemented with microfluidic systems. In this process, a patient sample is introduced into a microfluidic cartridge, which is then activated in an analyzer to perform nucleic acid amplification within the cartridge.

[0002] In the process of nucleic acid extraction to prepare for nucleic acid amplification, the so-called bind-wash elute (BWE) method involves first disrupting the cells in the sample under investigation using a suitable method, i.e., chemical, thermal, or mechanical lysis. This process also releases the nucleic acids from bacteria, viruses, or fungi in the sample. Since the samples are typically placed directly into a liquid transport medium after collection and mixed with this medium before being introduced into the microfluidic system, the transport media used, such as Copan eNAT™, can, depending on their chemical composition, support lysis and stabilize the released nucleic acids during transport and storage. Thus, the partially released nucleic acids are mixed with the remaining sample and the transport medium before being introduced into the cartridge.For a potentially more thorough lysis, a lysis buffer can be added to the sample in the cartridge.

[0003] Typically, the lysed sample in the cartridge is mixed with a binding buffer and, in a first step of the BWE procedure, brought into contact with a solid phase, particularly a silica filter, within the cartridge. The nucleic acid is adsorbed and retained by interaction with the surface of the solid phase, while the remaining mixture of sample, transport medium, binding buffer, and any lysis buffer is removed from the solid phase, particularly by the filter. This separation of the nucleic acids from other components, especially via a solid phase, is commonly referred to as purification. In suitable cases, the lysis buffer can be used at least as part of the binding buffer, since the chaotropic substances typically contained in the lysis buffer promote the binding of the nucleic acid to the solid phase, especially a silica filter.Following binding, any remaining interfering residues, such as cell debris, are removed from the solid phase using a suitable washing buffer, which is also considered part of the purification process. By selecting an appropriate elution buffer, the nucleic acids are eluted from the solid phase under suitable conditions, particularly at elevated temperature, and can then be used for subsequent amplification reactions, such as polymerase chain reactions or isothermal amplification techniques, which are generally referred to as nucleic acid amplification techniques (NAAT).

[0004] In the BWE method, the choice of lysis and binding medium is crucial, as these media come into contact with the typically liquid sample, and are mixed with it, thus influencing the chemical conditions of the environment surrounding the released nucleic acids. These conditions are essential for the successful binding of the nucleic acids to the solid phase. Chaotropic salts or alcohol-containing solutions, such as ethanol, can be used to create suitable chemical conditions for the selective purification of nucleic acids on the solid phase. However, these can hinder or completely prevent subsequent nucleic acid amplification. Therefore, it is essential that washing the nucleic acids bound to the solid phase also removes residues of the lysis reagents and binding buffer, particularly the chaotropic substances.

[0005] However, if the patient sample containing the biological cells is taken up in a non-lyseating transport medium, particularly one without lyse properties such as Copan UTM® or Amies medium, to keep the cells intact, the sample is first passed through a suitable solid phase, especially a filter, in the cartridge after infusion and before the nucleic acids are released. This allows the cells to be retained by size exclusion, particularly based on the selected pore size of the filter, and separated from the transport medium and other sample components. This is also known as the cell capture approach. A wash buffer placed upstream on the cartridge can also be used, which separates any remaining sample particles from the retained cells and removes them from the solid phase.The cells are then lysed with a lysis medium, which releases the nucleic acids contained within the cells and carries them away from the solid phase, in the case of a porous filter through the filter pores. The lysis medium, containing the released nucleic acids, is then mixed with reagents for nucleic acid amplification. These reagents, particularly a so-called master mix, are typically also included in the cartridge, for example, in the form of a bead. Since the lysis medium is directly incorporated into the reaction mixture, it must not contain any substances that could impair nucleic acid amplification.Therefore, a lysis medium with chaotropic substances, typically used for the BWE process and also referred to as a hard lysis medium, is fundamentally unsuitable for the cell capture process, and a detergent such as Octoxinol 9 (Triton®-X-100) is used as a lysis medium that is harmless for nucleic acid amplification and is also referred to as a soft lysis medium, as described, for example, in the German patent application DE 10 2020 203 035 A1. Disclosure of the invention Advantages of the invention

[0006] Against this background, the invention relates to a method for purifying nucleic acids from cells in a sample using a microfluidic device, for example as preparation for nucleic acid amplification, for example for the amplification and detection of nucleic acid sequences of pathogens in the sample.

[0007] A sample can be understood to mean, in particular, a biological sample, that is, a sample comprising a part of a living organism, especially a body fluid or a part of body tissue, for example, blood, sputum, urine, or a swab. Biological cells, in this context, refer specifically to cells of multicellular organisms such as mammals, including humans, or of single-celled organisms such as bacteria, but also viruses or fungi. The sample, in particular after being taken from a living organism, especially a patient, is placed in a medium for transport from the organism to the analysis, i.e., to a microfluidic device. This medium is referred to as the transport medium and is preferably suitable for preserving at least certain biological cells contained in the sample. Therefore, the term "sample" also refers to the mixture comprising the biological sample and such a transport medium.A transport medium for preserving at least certain biological cells contained in the sample, hereinafter also referred to as a stabilizing transport medium, is in particular a liquid transport medium for preserving microorganisms, especially pathogens in the form of bacteria, viruses, fungi, or other single-celled organisms. For example, depending on the type of microorganism to be preserved, the medium may be Amies medium, Stuart medium, or Copan Universal Transport Medium® (UTM®), or weak saline solutions based on sodium chloride or phosphate-buffered saline (PBS) solutions. According to particular embodiments of the invention, the transport medium may also comprise a nutrient medium, also called a culture medium, for preserving certain microorganisms.

[0008] The microfluidic device can in particular be a microfluidic cartridge for receiving and processing the sample, wherein the cartridge can be received into an analytical instrument for controlling the processing of the sample in the cartridge.

[0009] The sample, including the cells and transport medium contained within it, is introduced into the microfluidic device, particularly into a sample input chamber of the device. The mixture of sample and transport medium, and in particular the portion of this mixture introduced into the device, is hereinafter referred to simply as the sample. Cells in the sample are then lysed to release nucleic acids. This lysed sample, which in particular still contains the transport medium, is subsequently applied to a solid phase of the device to bind the released nucleic acids to the filter. In the case of a filter as the solid phase, in particular a silica filter, the lysed sample can be conveyed, in particular pumped, through the filter.After the nucleic acids bind to the filter, particularly after a predetermined time has elapsed following the application of the lysed sample to the solid phase or after pumping the lysed sample through the filter, the filter is preferably washed to remove residues from the sample, especially components such as chaotropic salts, and from the binding or lysis medium. The washing buffer used for this purpose is preferably located upstream of the device. Subsequently, the nucleic acids bound to the filter and now purified can be eluted, i.e., released from the filter, with an elution medium, particularly an elution buffer, especially for subsequent nucleic acid amplification.

[0010] Cell lysis can be carried out (bio)chemically by mixing with a lysis medium comprising chemical or enzymatic lysis reagents, mechanically, in particular by ultrasound, and / or thermally by heating the cells. Preferably, the sample is lysetated by adding a lysis medium to the sample, the lysis medium being preferably located upstream of the microfluidic device. The lysis medium is preferably an aqueous solution. Preferably, the lysis medium contains chaotropic substances, in particular at a concentration of 1.5 to 4.5 molars (M, molar being the unit for moles per liter). The chaotropic substances can be, in particular, chaotropic salts such as barium salts, guanidine hydrochloride, thiocyanates such as guanidinium thiocyanate, perchlorates such as sodium perchlorate, or sodium chloride.Preferably, the lysis medium, in particular the lysis buffer, does not contain any alcohol, in particular no ethanol and / or no propanol.

[0011] The binding of the nucleic acids released from the sample by lysis is supported by a binding medium added to the sample. This binding medium, which is preferably located upstream of the apparatus and is preferably an aqueous solution, can be a binding buffer that is added to the sample. According to a particular embodiment, the lysis medium can form part of the binding medium or be identical to it. For binding to the nucleic acids to the filter, the binding medium and / or the lysis medium preferably have a concentration of at least 1.5 molars of chaotropic salts, more preferably a concentration between 1.5 and 4.5 molars, and most preferably between 3.0 and 3.5 molars. For example, the binding medium and / or the lysis medium comprise guanidinium thiocyanate and / or guanidine hydrochloride and / or the other chaotropic salts mentioned above as the chaotropic salt.Preferably, the binding medium, in particular the binding buffer, contains no alcohol, especially no ethanol and / or no propanol. The lysis, the preferably addition of the lysis medium, and / or the addition of the binding medium preferably take place in the sample input chamber or in another chamber of the apparatus, preferably not in the chamber containing the filter for binding the nucleic acids.

[0012] The invention thus advantageously enables the release of nucleic acids located within intact cells in a sample, their binding to a solid phase of a microfluidic device, and their separation from other components of the sample. Such direct binding of the nucleic acids to the solid phase allows for thorough washing, leaving only negligible residues from the sample, the transport medium, and especially the binding or lysis medium on the solid phase. This further advantageously facilitates the amplification, particularly of portions of these nucleic acids, via PCR or isothermal amplification practically without inhibitory substances. The invention thus allows the use of a hard lysis medium and / or binding medium with chaotropic reagents, instead of the previously used soft, detergent-based lysis buffer, even for samples containing intact cells in stabilizing transport media.While in the cell-capture approach, due to insufficient binding conditions for nucleic acids, only intact cells can be retained and washed on the solid phase before lysis occurs, the invention allows for the removal of remnants of the lysed cells, particularly cell fragments and debris, during purification, thanks to the lysis occurring before contact with the solid phase. This results in a more thorough purification. Furthermore, the invention enables a higher yield of nucleic acids through binding to the solid phase. In the cell-capture approach, some cells are damaged or ruptured during sample processing before lysis, releasing nucleic acids that are then not retained by the filter during washing.Furthermore, it is particularly advantageous that nucleic acids from microorganisms with smaller diameters, such as viruses or microorganisms without a (robust) cell wall, such as mycoplasmas, can also be isolated from the sample, which cannot be retained in a well-defined manner by a filter in the cell-capture approach.

[0013] Furthermore, a particular advantage is that the method is also applicable to samples taken in non-stabilizing, inactivating, or even lysing transport media such as Copan eNAT™ or Roche cobas® PCR medium. Due to the lack of cell-stabilizing or lysing effect, at least some of the cells in the sample will be disrupted and the nucleic acids contained within them released, but this is not critical for the further course of the procedure. Thus, the method can be used with a wide variety of transport media and is effective with all commonly used transport media for such samples.When a lyses transport medium such as Copan eNAT™ is used for sample transport, the cell lysis step is at least partially performed by mixing the sample with the transport medium before it is taken up into the microfluidic device. To ensure thorough lysis and promote binding to the solid phase, an additional lysis step can also be performed within the device after the sample has been taken up, particularly by adding a lysis medium to the sample.

[0014] To further support the binding of nucleic acids to the filter, the binding medium and / or the lysis medium can contain substances to lower the pH, in particular buffer systems to adjust the pH to between 4.0 and 8.0, preferably between 5.0 and 7.0. If the mixture of sample, (preferably stabilizing) transport medium, and lysis and / or binding buffer that comes into contact with the solid phase, especially the filter, has a pH of less than 7, preferably between 5 and 7, the binding of the nucleic acids is significantly supported, particularly with a silica filter.

[0015] According to a particularly advantageous embodiment of the invention, a processing medium is added to the sample. It was recognized during the course of the invention that the addition of a processing medium can significantly enhance the chaotropic effect of the binding or lysis medium, particularly in transport media containing substances with cosmotropic properties. The cosmotropic effect present in such a processing medium, especially due to cosmotropic salts in the transport medium, can advantageously be selectively reduced. For this purpose, the processing medium preferably comprises a complexing agent containing ammonium ions, which forms complexes with alkaline earth ions.

[0016] The processing medium may contain, in particular as a complexing agent, at least one salt selected from the group consisting of ammonium hydrogen citrate, diammonium hydrogen citrate, ammonium citrate, ammonium thioglycolate, ammonium chloride, ammonium acetate, tetramethylammonium citrate, and tetramethylammonium thioglycolate. The processing medium may, in particular, include a complexing agent disclosed in German patent application DE 10 2020 203 035 A1. The content of German patent application DE 10 2020 203 035 A1, filed on March 10, 2020, and published on September 19, 2021, is incorporated as part of this disclosure.Such a complexing agent further has the advantage that, by adjusting the chemical environment, the solubility product of suspended particles in the sample, particularly those originating from a transport medium such as Amies, can be shifted in such a way that the suspended particles dissolve and, during the filtration step, pass through the solid phase, especially the silica filter, without settling on it. The processing medium thus advantageously supports the use of the presented uniform and practically universal method for purifying nucleic acids from samples in stabilizing and non-stabilizing transport media by facilitating the use of a hard buffer from the BWE method and reducing or even eliminating disadvantages of the cell capture method.

[0017] The processing medium, which is preferably located upstream of the cartridge, can be added to the sample before or after the addition of the lysis medium. The dilution effect can be precisely controlled by selecting the order and quantity of the processing and lysis mediums added. Particularly with regard to the effect of chaotropic substances, the addition of the processing medium can have two competing effects, the extent of which can be controlled by the concentration of the aforementioned complexing agents in the processing medium: on the one hand, compensation of the effect of cosmotropic substances, and on the other hand, a reduction of the chaotropic effect through dilution. According to a particular embodiment, the processing medium forms part of the binding medium or the lysis medium.For example, the processing medium can be mixed with the binding medium and / or the lysis medium before the resulting mixture is added to the sample. Alternatively, the lysis medium can be added to the sample first. Only after lysis has occurred, particularly after a predetermined time, is the mixture of sample and lysis medium, or a portion thereof, mixed with the processing medium and the binding medium. This is particularly advantageous if lysis without the processing medium is sufficient or even more effective, and if the processing medium and the binding medium are compatible, for example, already present as a single medium, and preferably also compatible with the lysis medium, to establish favorable binding conditions after mixing the three media.

[0018] The invention also relates to a microfluidic device, in particular configured for carrying out the method according to the invention, wherein a binding medium, a lysis medium and / or a processing medium are positioned upstream in the device such that they can be mixed with a sample introduced into a sample input chamber of the device before the sample is applied to a solid phase, in particular to a filter of the device. As described above, the device, which can in particular be configured as a microfluidic cartridge, can each have a chamber for pre-storing the binding medium, lysis medium and / or processing medium.

[0019] According to a preferred embodiment of the invention, the binding medium, the lysis medium, and / or the processing medium are pre-positioned in the device as a common combined medium, particularly in the form of a buffer, for example, in liquid form (e.g., as a reagent bar) or in dried form, in a reagent chamber of the device or cartridge. Pre-positioning the binding medium and the processing medium creates an effectively universal device, particularly a cartridge, which is suitable for all commonly used transport media, whether lyses or cell stabilizers, and can purify nucleic acids from samples in these transport media.

[0020] For example, the device includes a combined medium which, as already described above, performs and thus replaces the functions of the lysis medium, the binding medium, or alternatively, the binding medium and the processing medium. Preferably, the combined medium assumes the functions of the binding medium, the lysis medium, and the processing medium. For example, the combined medium is produced by mixing lysis buffer, binding buffer, and processing buffer. Such a combined medium further supports the device's versatility with respect to transport media as described above.

[0021] The term "medium," particularly in the case of lysis medium, binding medium, processing medium, or combination medium, can refer to a solution, especially an aqueous solution. The term "buffer," particularly in the case of lysis buffer, binding buffer, or processing buffer, can, in a narrower sense, refer to the chemical concept of a buffer, specifically a mixture whose pH (concentration of hydronium ions) changes significantly less upon the addition of an acid or a base than would be the case in an unbuffered system. Alternatively, and more generally, such a buffer can also be understood as a solution, especially an aqueous solution. Brief description of the drawings

[0022] Exemplary embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. The same reference numerals are used for the elements shown in the various figures that have a similar effect, thus omitting a repeated description of the elements.

[0023] They show Fig. 1 a flowchart of an embodiment of the method according to the invention as well as Fig. 2 an embodiment of the microfluidic device according to the invention. Embodiments of the invention

[0024] Fig. Figure 1 shows a flowchart for an embodiment of the method 500 according to the invention.

[0025] In a first step, a biological sample is prepared for testing for the presence of pathogens using a PCR test in a microfluidic point-of-care molecular diagnostics system. The biological sample, which could be, for example, a swab, sputum, or blood sample from a patient, is transferred directly into a transport medium after collection. This can be done, for example, via a flocked swab into a container containing the transport medium, such as using the Copan eSwabs™ (Copan Liquid Amies Elution Swab) swab and transport system. The transport medium includes a stabilizing agent to preserve the cells of potential pathogens in the sample.For example, depending on the type of microorganism to be detected, the transport medium may be an Amies medium or UTM® or a weak saline solution such as 0.9% sodium chloride solution or PBS.

[0026] The mixture comprising the biological sample and the transport medium comprising the transport medium, hereinafter collectively referred to as the sample, are introduced in a second step 502 into a microfluidic device, for example a few hundred microliters of this mixture, which is hereinafter also referred to as the sample.

[0027] Fig. Figure 2 schematically shows an excerpt of the structure of a microfluidic cartridge 100. The cartridge 100 can, in particular, comprise a layered structure with a fluid layer encompassing the majority of the entire fluidic network of the cartridge, a pneumatic layer, and a stretchable membrane layer arranged between the fluid layer and the pneumatic layer. Depending on the process, the pressure applied via a pneumatic interface causes the membrane to expand in specific locations within chambers of the fluidic and pneumatic layers in order to move fluids and reagents within the cartridge. The sample 10 is introduced into a sealable sample entry chamber 101.

[0028] In a third step 503, the cells in the sample are lysed by adding a lysis medium 111 to release the nucleic acids from the cells and obtain a lysed sample. The lysis medium 111 is located upstream of the sample input chamber 101 in a first reagent chamber 110, which is fluidically connected to the sample input chamber 101, for example in the form of a reagent latch that can be opened by mechanical force. It can be conveyed into the sample input chamber 110, for example by appropriate localized deflection of the stretchable membrane layer, and mixed with the sample 10 for cell lysis.

[0029] In this example, the lysis medium 111 comprises a lysis buffer with chaotropic substances at a concentration between 1.5 and 4.5 molar, for example, 3.7 molar. The chaotropic substances can be chaotropic salts, such as guanidinium thiocyanate, for example, as a 1:1 composition of thiocyanic acid and guanidine, with a proportion between 25 and 40 wt% in an aqueous solution.

[0030] Preferably, a processing medium 121 is added to sample 10 beforehand, for example as a processing buffer, which may be located upstream in a second reagent chamber 120 and contains at least one salt as a complexing agent selected from the group consisting of ammonium hydrogen citrate, diammonium hydrogen citrate, ammonium citrate, ammonium thioglycolate, ammonium chloride, ammonium acetate, tetramethylammonium citrate, and tetramethylammonium thioglycolate. The processing medium 121 can at least partially reduce any cosmotropic effects introduced into sample 10 by the transport medium and thus indirectly preserve the chaotropic effect of the lysis buffer 111 added to the sample. By adjusting the concentrations of the components in the processing medium 121 and the lysis buffer 111 in light of the composition of the culture medium used, an effective operating point for the lysis of the cells in the sample can be established.Alternatively, the processing medium 121 can be added to the sample only after the lysis medium 111 has been added. Particularly in other configurations, especially depending on the desired operating point, the addition of the processing medium can be omitted entirely.

[0031] The mixing of sample 10 with the lysis buffer 111 and / or with the processing medium 121 can take place in the sample input chamber 101.

[0032] In a fourth step 504 of the process 500, a binding buffer 131, located upstream in a third reagent chamber 130, is added to the sample, for example, also by conveyance into the sample input chamber 101, in order to establish suitable conditions for the binding of the nucleic acids released in the sample to a filter. For example, the filter can be a silica filter 161 based on a porous silica membrane, such as POREX®, and in the form of a filter frit, located in a further chamber 160, called filter chamber 160. For the establishment of the binding bonds, the binding buffer 131 can contain chaotropic substances, in particular chaotropic salts.To adjust these binding bonds, the binding buffer 131 can be formulated in a way that is compatible with the other buffers 111 and 121, such that the mixture resulting from the mixing of the binding buffer 131 with the sample 10 and preferably the lysis buffer 110 and / or the processing medium 121 has a concentration of the chaotropic salts in the range of 1.6 to 2.2 molars. The addition of the processing medium 121 described above can lower the concentration of the chaotropic substances or salts, for example from 3.7 molars to 1.5 M, in order to achieve adequate, but not excessive, binding of the nucleic acids to the filter 161 and simultaneously reduce the influence of the chaotropic substances on the subsequent steps.To promote bond formation, substances that lower the pH can be added to the lysis, binding, or processing medium, for example, to give the mixture coming into contact with the solid phase 161 a pH between 5 and 7. These substances can be, for example, a citric acid / citrate buffer, an acetic acid / acetate buffer, and / or a thioglycolic acid / thioglycolate buffer. These buffers are particularly suitable for adjusting the pH to the range of 5 to 6. If a citric acid / citrate buffer is to be used, the complexing agent used as the processing medium is preferably ammonium hydrogen citrate, ammonium citrate, or tetramethylammonium citrate, to which citric acid is additionally added.If the buffer is to be an acetic acid / acetate buffer, the complexing agent is preferably ammonium acetate to which acetic acid is added. If a thioglycolic acid / thioglycolate buffer is to be used, the complexing agent is preferably ammonium thioglycolate or tetramethylammonium thioglycolate to which thioglycolic acid, also known as mercaptoacetic acid, is added. It is also possible, in principle, for the complexing agent to contain only one of the salts suitable for buffer formation—that is, a hydrogen citrate, a citrate, an acetate, or a thioglycolate—and for the corresponding acid to be released from it by the addition of another organic acid. Mandelic acid is particularly suitable for this purpose. The addition of another organic acid, such as mandelic acid, to an existing system of salt and corresponding acid is also possible.

[0033] The binding buffer 131 can, as described above, be a combined binding and lysis buffer, i.e., a buffer that both lyses the cells in the sample and creates suitable chemical conditions for binding the released nucleic acids to the filter 161. For example, such a combined buffer, which is preferably also located upstream of the cartridge in one of the reagent chambers 110, 130, has a guanidinium thiocyanate concentration of between 2.5 and 3.5 molars and a pH value between 6 and 7. Particularly due to space constraints, a common medium or combined medium, especially a common buffer, can be used instead of the individual media and is preferably located upstream of the cartridge in one of the reagent chambers 110, 120, 130, wherein the common medium provides the functions of lysis medium, binding medium, and processing medium.The common medium can be prepared, for example, by mixing lysis buffer, binding buffer, and processing buffer, either outside the cartridge or inside the cartridge, for example, only when the procedure is carried out by mixing in one of the reagent chambers 110, 120, 130.

[0034] After a predetermined time, for example a few seconds to a few minutes, the sample, adjusted to the specified binding conditions, is conveyed in a fifth step 505 onto the filter 161 in the filter chamber 160, which is fluidically connected to the sample input chamber 101. Preferably, this fluidic connection can be separated by a valve 162. For example, the application to the filter 161 can be accomplished by pumping the sample 10 through the filter 161 into a waste chamber 170 of the cartridge 100.

[0035] In a sixth step 506 of the process 500, the filter 161 with the nucleic acids bound to it is washed with a washing buffer 141 located upstream in a fourth reagent chamber 140 of the cartridge 100 in order to remove residues from the sample, in particular components, for example chaotropic salts, of the binding or lysis medium from the filter. The washing buffer can be a buffer or solution commonly used for the purification of nucleic acids, preferably without ethanol, and preferably containing less than 0.1 mol per liter of chaotropic substances, in particular chaotropic salts. For example, the washing buffer comprises a polyethylene glycol / water mixture.

[0036] In a seventh step 507 of the process 500, an elution buffer 151 is used as an elution medium to break the binding conditions and thus release the nucleic acids from the filter 161. The elution buffer 151 can be located upstream of the filter chamber 160 in a fifth reagent chamber 150, which is fluidically connected to the filter chamber 160. The elution buffer can be a typical buffer for dissolving nucleic acids from silica, for example, distilled or buffered water, possibly with surfactants such as polysorbate 80 (Tween® 80).

[0037] In a preferred eighth step 508, further processing of the eluted nucleic acids can take place, for example, amplification of portions of the nucleic acids using a (quantitative real-time) polymerase chain reaction, isothermal amplification, or ligase chain reaction in a further chamber 180 (hereinafter referred to as analysis chamber 180) for the detection of the nucleic acids and thus associated pathogens in the sample. Analysis chamber 180 may, for example, contain a PCR master mix in the form of a PCR bead 181. Direct mixing of the elution buffer 151 with the nucleic acids it contains is unproblematic, since no or negligible chaotropic substances are present that could hinder amplification.

[0038] The cartridge 100 can have a further chamber as a detection chamber 190, in which, for example, a microarray 191 with immobilized probes for hybridizing the sought and amplified nucleic acid segments can be arranged, which can then be read out, for example, optically via fluorescence spectroscopy.

[0039] As explained above, this method can be used for 500 and especially for those who... Fig. The microfluidic device 100 described in section 2 can also be used for samples which are contained in non-stabilizing, inactivating or even lysing transport media such as Copan eNAT™ or Roche cobas® PCR medium. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] DE 10 2020 203 035 A1 [0005, 0016]

Claims

Method (500) for purifying nucleic acids from cells in a sample (10) with transport medium, in particular for nucleic acid amplification, using a microfluidic device (100), comprising the following steps:• Incorporating (502) a sample (10) into the microfluidic device (100), wherein the sample (10) comprises cells with nucleic acids contained therein and wherein the sample (10) comprises a transport medium for the cells, in particular a stabilizing transport medium.• Lysing (503) the cells in the sample (10), in particular by adding a lysis medium (111), in order to release the nucleic acids from the cells and obtain a lysed sample (10).• Applying (505) the lysed sample (10) to a solid phase (161) of the device (100), in particular a filter (161), wherein the lysed sample (10) comprises a binding medium (131) for binding the released nucleic acids to the filter (161). • Preferably washing (506) the filter (161) in order to remove residues from the sample (10), in particular components, for example chaotropic salts, of the binding or lysis medium (111), from the filter (161). • Preferably eluting the nucleic acids bound to the filter (161) with an elution medium (151). Method (500) according to claim 1, wherein the lysing (503) of the sample (10) comprises adding a lysis medium (111) to the sample (10), wherein the lysis medium (111) is preferably stored upstream in the microfluidic device (100). Method (500) according to claim 2, wherein the lysis medium (111) forms at least a part of the binding medium (131). Method (500) according to one of the preceding claims, wherein the binding medium (131) and / or the lysis medium (111) for a binding effect for binding the nucleic acids to the filter (161) have a concentration of at least 1.5 molar of chaotropic salts, preferably a concentration between 1.5 and 4.5 molar. Method (500) according to one of the preceding claims, wherein the binding medium (131) and / or the lysis medium (111) for a binding effect for binding the nucleic acids to the filter (161) comprise substances for lowering the pH value when mixed with the sample (10). Method (500) according to one of the preceding claims, wherein the lysis medium (111) and / or the binding medium (131) comprise guanidinium thiocyanate or guanidine hydrochloride as chaotropic salt. Method (500) according to one of the preceding claims, wherein a processing medium (121) is added to the sample (10), preferably to support a chaotropic effect of the binding medium (131) and / or lysis medium (111). Method (500) according to claim 7, wherein the processing medium (121) forms part of the binding medium (131) or lysis medium (111). Method (500) according to claim 7 or 8, wherein the processing medium (121) contains as complexing agent at least one salt selected from the group consisting of ammonium hydrogen citrate, di-ammonium hydrogen citrate, ammonium citrate, ammonium thioglycolate, ammonium chloride, ammonium acetate, tetramethylammonium citrate and tetramethylammonium thioglycolate. Method (500) according to one of the preceding claims, wherein the lysing (503) of the cells, in particular the addition of the lysis medium (111) and / or the addition (504) of the binding medium (131) takes place in a chamber other than the chamber comprising the filter (161), for example in a sample input chamber (101) of the microfluidic device (100). Method (500) according to one of the preceding claims, wherein the sample (10) comprises an inactivating, in particular lysing, transport medium for the cells, and wherein the lysing (503) of the cells in the sample (10) takes place at least partially before the uptake (502) of the sample (10) into the microfluidic device (100) by the transport medium. Microfluidic device (100), in particular configured to carry out one of the methods (500) according to one of the preceding claims, wherein a binding medium (131), a lysis medium (111) and / or a processing medium (121) are disposed upstream in the device (100) in such a way that they can be mixed with a sample (10) introduced into a sample introduction chamber (101) of the device (100) before the sample (10) is applied to a solid phase, in particular to a filter (161), of the device (100). Microfluidic device (100) according to claim 12, wherein the binding medium (131), the lysis medium (111) and / or the processing medium (121) are stored as a common combination medium in the device (100).

Images