DE102015215894A1 - Method of enriching biomolecules and removing biomolecules from a biological sample - Google Patents

Abstract

The invention relates to processes for the enrichment of biomolecules and for the removal of biomolecules from a biological sample, characterized in that - in the presence of particles - a biological sample is mixed with an alginate solution and with salts of di- or polyvalent cations or with an acid in which an alginate gel-biomolecule complex forms on the particles, which is removed by separation of the particles from the sample and from which subsequently the biomolecules or constituents of the biomolecules are released. The biomolecules to be enriched include cell-free nucleic acids, viruses or subcellular microparticles. The method is an improved and simpler method than the method described in the patent DE 10 2008 023 297 B4.

Description

The invention relates to a simple method for enriching biomolecules and for removing (isolating) the biomolecules from a sample for a subsequent investigation of these biomolecules or for further processing. The biomolecules to be enriched include cell-free nucleic acids, viruses, or subcellular microparticles (eg, exosomes). The process is an improved and simpler process than that in the patent DE 10 2008 023 297 B4 described method.

It is known that free-circulating nucleic acids, exosomes and, in the case of a viral infection, also virus particles are present in cell-free body fluids. All of these different biomolecules are of great importance for the diagnosis of diseases. In particular, cell-free nucleic acids and exosomes play an increasingly important role. All these biomolecules also have in common that they are present only in very low concentrations in body fluids. This complicates their diagnostic use. The only way out is to process larger sample volumes to finally have available a sufficient amount of biomolecules. As important starting samples are e.g. Body fluids such as blood plasma or serum or urine in question.

There are currently only a few methods that make it possible to enrich or isolate these biomolecules from a large-volume sample or subsequently to use the enriched biomolecules for the extraction of nucleic acids.

For example, for the enrichment of exosomes from a biological sample, there is the use of ultracentrifugation techniques. This leads to the accumulation of exosomes at the bottom of the reaction vessel. This method is bound to an ultracentrifuge and also time consuming and unsuitable for routine diagnostics. Furthermore, the technology of ultrafiltration is used. Again, this method is time consuming and very expensive. Alternative methods consist of immunoprecipitation of the exosomes by means of an immunoplate or by means of immunobeads. This type of enrichment of the exosomes is time-consuming and prone to failure due to the reagents to be used and expensive. In addition, only 200-500 μl of sample can be used with this technology. Ultracentrifugation techniques can also be used for the accumulation of viruses. Alternative methods consist in the precipitation of virus particles by means of polyethylene glycol / sodium chloride and a subsequent centrifugation ( Yamamoto et al., Virology 40 (1970) 734; Morandi et al., J. Clin. Microbiol. 36 (1998) 1543-1538 ). It uses different mixtures of PEG and sodium chloride and mixes these reagents with the biological sample. Subsequently, the batch is incubated for a long time in the cold and then the virus (protein) -NaCl / PEG precipitates recovered by centrifugation. These methods are complicated and take a lot of time. Furthermore, the further processing of the precipitates for the isolation of the viral nucleic acids is problematic. Often, the precipitates are very difficult to bring back into solution. This significantly affects the efficiency and quality of the nucleic acid isolation. The patent DE 19856415 C2 describes a method which makes use of the known NaCl / PEG precipitation, wherein subsequently the isolation of the nucleic acids is realized in a manner known per se via the binding to a silicate-like solid phase. The extent to which this method differs from the well-known method of NaCl / PEG precipitation with the known problems is not apparent. In addition, this method also requires a cold incubation and a twenty-minute centrifugation. The method should allow to isolate viral nucleic acids from a sample of up to 10 ml. Another commercially available variant, which is to allow the processing of up to 1 ml of sample based on the accumulation of viral nucleic acids using a special detergent. An initial incubation of the sample with a lysis reagent leads to lysis of the viruses. Subsequently, it comes to the formation of a "detergent-nucleic acid complex". The batch is centrifuged and the resulting pellet subsequently proteolytically treated and the nucleic acid in turn isolated in a conventional manner via the attachment to a silicate solid phase (QIAamp UltraSens Virus Handbook). Again, it is pointed to problems with the resuspension of the pellet. In addition, the method also allows only the processing of samples with a volume of a maximum of 1 ml.

The procedure for isolating circulating cell-free DNA from large-volume samples is to scale up all the buffer components needed for the extraction. The skilled worker is aware that this is associated with an enormous amount of reagents and time.

It will also be apparent to those skilled in the art that for different biomolecules (e.g., viruses, or DNA or subcellular particles), different methods are always needed to concentrate the biomolecules from a sample.

A completely new approach for the concentration of biomolecules from a biological sample and for the subsequent extraction of nucleic acids is described in the patent DE 10 2008 023 297 B4 disclosed.

The method is based on the use of polysaccharide derivatives for complexing the biomolecules contained in a sample. The polysaccharide derivatives are the salts of polyuronic acids. Particularly suitable are the so-called. Alginates of alginic acids. Alginates are structural elements of brown algae. Alginate is a polysaccharide consisting of 1,4-linked α-L-guluronic acid (G) and β-D-mannuronic acid (M).

It forms homopolymeric regions in which mannuronic acid or guluronic acid is present as a block.

Alginates are used in the food, pharmaceutical and cosmetics industries, e.g. as a gelling agent in the food industry, as Apreturmittel for textiles, for the production of photographic papers or in the dental practice for the production of dental and Kieferabformungen.

The patent describes the use of the ability of alginates to gel in low calcium solutions and to form so-called hydrogels. The cause of the gelation is due to the incorporation of calcium ions in the zigzag structure of the GG blocks. The zigzag structure of another alginate molecule is then deposited on this zone. This leads to the formation of three-dimensional structures. The formation of gels is also in combination with strong acids. In addition, the resulting gel structures can also be specifically destroyed again.

Taking advantage of the formation of alginate gels, the accumulation of biomolecules can be done easily and quickly and subsequently the isolation of nucleic acids can be carried out.

• 1. Mischen einer wässrigen Alginatlösung mit einer flüssigen biologischen Probe
• 2. Zugabe einer wässrigen Lösung, welche die Gelausbildung/ Pelletbildung induziert (z.B. Verwendung einer 1 M Kalziumchloridlösung oder einer 1%igen Salzsäurelösung)
• 3. Mischen der Probe und kurze Inkubation bei Raumtemperatur
• 4. Zentrifugation der Probe und Entfernung des Überstandes
• 5. Auflösen des Pellets oder Gelstückchens und damit Freisetzung der Biomoleküle und ggf. nachfolgende direkte Isolierung von DNA oder RNA in an sich bekannter Art und Weise

The procedure is described as follows:

• 1. Mixing an aqueous alginate solution with a liquid biological sample
• 2. Addition of an aqueous solution which induces gel formation / pellet formation (eg using a 1 M calcium chloride solution or a 1% hydrochloric acid solution)
• 3. Mix the sample and briefly incubate at room temperature
• 4. Centrifugation of the sample and removal of the supernatant
• 5. Dissolution of the pellet or Gelstückchens and thus release of the biomolecules and optionally subsequent direct isolation of DNA or RNA in a conventional manner

The process disclosed in the patent is extremely efficient and makes it possible to quickly and easily use even large-volume samples for the enrichment of biomolecules.

However, the method described has a great disadvantage. Automation of the process is difficult to perform. This is due to the fact that the enrichment step must always be done by centrifugation. As is known, the integration of a centrifugation step into an automated process is not easy to implement and, moreover, significantly increases the cost of a machine.

Object of the invention

The invention had the object to eliminate the disadvantages of the known technical solutions.

Solution of the task

The problem has been solved according to the features of the claims. The method according to the invention makes it possible to enrich biomolecules (also from large-volume samples) and subsequently release the biomolecules or, if appropriate, isolate nucleic acids (DNA and RNA) or release the nucleic acids from these.

The method makes it possible to quickly and easily implement all necessary process steps in an automated process. Surprisingly, this can be done in a very simple way - without a centrifugation step.

The method is based on the known method, a biological liquid sample with an alginate solution and with salts of di- or polyvalent cations (eg, calcium, zinc, - or aluminum salts) or with an acid to put. However, the enrichment of the biomolecules is not carried out subsequently by a centrifugation step. Particles are added to the sample, preferably magnetic or paramagnetic particles. It does not matter what kind of particles it is. Both the size of the particles, as well as their functionalization is of minor importance. It can therefore be used nanoparticles, microparticles or larger particles. Magnetic particles are best suited for easy separation of the particles. In this case, the particles are separated by means of a magnet and the sample supernatant is removed. After removal of the sample supernatant, the biomolecules to be enriched (subcellular particles, viruses or nucleic acids) are completely in a complex of alginate gel and magnetic or paramagnetic particles. This enrichment was thus surprisingly without the in the patent DE 10 2008 023 297 B4 listed centrifugation. The release of the complexed biomolecules can now be done by adding reagents that destroy the alginate gel structure again and thus release the complexed biomolecules. This can be done for example by means of a buffer solution containing a chelating agent (EDTA) or else by means of the addition of a solution of tri-sodium citrate dihydrate. After addition of these reagents, a brief resuspension of the particles and an incubation. The incubation serves the dissolution of the alginate gel structure and the release of the biomolecules. Subsequently, the magnetic or paramagnetic particles are separated by applying a magnetic field. The resulting supernatant contains the biomolecules, which can then be worked up or analyzed.

Thus, the process requires no Zentrifugationsschritt and can be extremely easily carried out completely automated. This makes it possible for the first time to process even large sample volumes efficiently and quickly.

The inventive method shows yet another advantage. In a specific embodiment, it is possible to select the particles used for the separation of the type that are capable of binding nucleic acids. This means that the particles serve, on the one hand, for the separation of the biomolecule-alginate complex and subsequently also for the extraction of high-purity nucleic acids. This method is particularly important when so-called circulating cell-free nucleic acids are to be enriched from a cell-free sample and subsequently isolated. For this purpose, an alginate solution and an aqueous solution containing salts of di- or polyvalent cations (eg calcium chloride or aluminum chloride) or a weak acid are added to the sample (serum, plasma, urine etc.) as well as paramagnetic or magnetic particles. These particles are selected to be able to bind nucleic acids under specific conditions. Such particles are known in the art and are used in the routine for the isolation of nucleic acids. After addition of these components, the mixture is mixed briefly and incubated for a few minutes at room temperature. Subsequently, the particles are separated by means of a magnet and the sample supernatant is removed. After removal of the supernatant, the particles (the alginate-gel particle complex) are treated with a buffer which allows destruction of the alginate-gel complex. For this, the particles are resuspended and the batch is incubated for a few minutes. In this case, other additives such as proteolytic enzymes, etc. may be added to the buffer. The incubation can be carried out at room temperature or at higher temperatures, preferably 50 ° C-70 ° C. After release of the complexed cell-free nucleic acid, this binds to the magnetic or paramagnetic particles. This can be done by the buffer used. Preferably, the binding of the DNA by the addition is made more effective a binding buffer. As a buffer reagent combinations can be used, those skilled in the patent DE 10 2008 023 297 B4 are known. Importantly, the first buffer allows destruction of the alginate gel structure and subsequent attachment of the released DNA, alone or in combination with an additional binding buffer.

It is also possible to use a binding buffer in addition to the lysis buffer, which allows to achieve a selectivity with respect to the nucleic acids to be purified (size fractionation or separation of DNA and RNA). The further extraction process is known to the person skilled in the art. The nucleic acids bound to the particles are washed and the nucleic acids are finally detached from the particles by adding water or by adding a low salt buffer.

By means of the method according to the invention, the entire process, starting with the enrichment of biomolecules up to the final extraction of highly pure nucleic acids, can thus also be carried out automatically. There is no need for a centrifugation step anymore. This represents a decisive advantage over that in the patent DE 10 2008 023 297 B4 disclosed method. In addition, by means of the method according to the invention, each sample volume can de facto be processed and is therefore not limited to small volumes and, moreover, it is easy to automate. The volumes of the liquid starting samples can be chosen arbitrarily, which allows a very wide range of applications. It can be used for example with samples of 500 .mu.l or even 10 ml. This is an enormously wide range of applications.

The method according to the invention is also suitable for generally isolating nucleic acids from a sample. If the nucleic acids to be isolated are not present as free nucleic acids, the sample can be lysed in a manner known per se. The nucleic acids released thereby from the sample are then in turn isolated according to the method according to the invention. Here, too, after dissolving the alginate gel structure (which contains the enriched nucleic acid), a "classical" purification of the nucleic acids can be carried out or, after the alginate gel structure has been dissolved by means of a basic low-salt buffer, the resulting solution can be used directly for a PCR reaction.

The invention will be explained in more detail with reference to embodiments, wherein the embodiments do not limit the process of the invention.

Exemplary embodiment 1: Enrichment of cell-free DNA from a plasma sample of 1 ml. Detection of the necessary combination of alginate solution, reagent for forming an alginate gel and particles separable by means of a magnetic field

The initial sample for the enrichment was human plasma. The plasma sample was again centrifuged for 10 min prior to use in order to remove any remaining cells. Further work was done with the supernatant. Three different methods were tested.

Sample 1: 30 μl of a 0.5% alginate solution was added to the sample and the batch was mixed briefly. Thereafter, 150 μl of a 1 molar calcium chloride solution and 50 μl of a magnetic particle suspension (MAG suspension, Analytik Jena AG) were added.

Sample 2: To the sample was added 30 μl of a 0.5% alginate solution and the batch was mixed briefly. Thereafter, 50 μl of a magnetic particle suspension (MAG Suspension, Analytik Jena AG) were added.

Sample 3: To the sample was added 150 of a 1 molar calcium chloride solution and the batch was mixed briefly. Thereafter, 50 μl of a magnetic particle suspension (MAG Suspension, Analytik Jena AG) were added.

The samples were then processed as follows.

The batch was mixed briefly and incubated for 10 minutes. Subsequently, the separation of the magnetic particles by means of a magnet. The supernatant was removed and the magnetic particles were washed with 1 ml of water. After renewed separation of the magnetic particles, the supernatant was completely removed.

400 μl buffer (4 M guanidine thiocyanate, EDTA) and 20 μl proteinase K (20 mg / ml) were added to the complex magnetic particle alginate gel DNA and the batch was resuspended with a pipette. This was followed by incubation for 15 min at 70.degree.

This step served to destroy the particle alginate gel-DNA complex and thus release the complexed DNA. This was followed by the addition of 400 .mu.l of a binding buffer (isopropanol / Triton X-100) and a remixing of the particles and an incubation for 2 min. This step served to bind the DNA to the particles. Thereafter, the magnetic particles were separated and the supernatant was discarded. The particles were subsequently washed with alcoholic washing buffers known to the person skilled in the art and finally dried. In the last step, the bound DNA was removed from the particles by adding 50 μl of H ₂ O and transferred to a new reaction vessel.

Reaktionsansatz Pro Probe: sense Primer (50 pmol/ µl) 0,1 µl antisense Primer (50 pmol/ µl) 0,1 µl Sonde (25 pmol/ µl) 0,1 µl dNTP-Mix (12,5 mM) 0,3 µl 10X PCR-Buffer (MgCl₂ included) 1,5 µl Taq-DNA-Polymerase 0,75 U PCR-Grade H₂O add 15 µl Amplifikations-/ Hybridisierungskonditionen Schritt 1: Denaturierung 95°C 120“ Schritt 2 Amplifizierung 45 Zyklen 95°C 4“ (Messung) 65°C 45“ The detection of the enrichment and subsequent extraction of cell-free DNA was carried out by means of real-time PCR. To this end, a human-specific target sequence estrogen receptor 1 was amplified. Real time PCR protocol for amplifying the human-specific target sequence (estrogen receptor 1).

Reaction batch per sample: sense primer (50 pmol / μl) 0.1 μl antisense primer (50 pmol / μl) 0.1 μl Probe (25 pmol / μl) 0.1 μl dNTP mix (12.5 mM) 0.3 μl 10X PCR Buffer (MgCl ₂ included) 1.5 μl Taq DNA polymerase 0.75 U PCR grade H ₂ O add 15 μl Amplification / Hybridization Conditions Step 1: denaturation 95 ° C 120 " step 2 amplification 45 cycles 95 ° C 4 " (Measurement) 65 ° C 45 "

Result PCR

The results in 1 show that only the combination of alginate solution, reagent for the formation of an alginate gel and particles separable by means of a magnetic field allow enrichment and subsequent extraction of DNA (black curves). This proves that the first step is the complexation of the free DNA with the alginate gel that forms and the added particles combine with this complex. This can then be separated by the application of a magnetic field of the particle alginate gel-DNA complex. A centrifugation, as in the patent DE 10 2008 023 297 B4 is described, is therefore not required.

Example 2: Enrichment of cell-free DNA from a plasma sample of 1 ml and 5 ml and subsequent extraction of the DNA

The initial sample for the enrichment was human plasma. The plasma sample was again centrifuged for 10 min prior to use in order to remove any remaining cells. Further work was done with the supernatant.

The 1 ml sample was treated as follows. 30 μl of a 0.5% alginate solution was added to the sample and the batch was briefly mixed. Thereafter, 150 μl of a 1 molar calcium chloride solution and 50 μl of a magnetic particle suspension (MAG suspension, Analytik Jena AG) were added. The batch was mixed briefly and incubated for 10 minutes. Subsequently, the separation of the magnetic particles by means of a magnet. The supernatant was removed and the magnetic particles were washed with 1 ml of water. After renewed separation of the magnetic particles, the supernatant was completely removed.

400 μl buffer (4 M guanidine thiocyanate, EDTA) and 20 μl proteinase K (20 mg / ml) were added to the complex magnetic particle alginate gel DNA and the batch was resuspended with a pipette. This was followed by incubation for 15 min at 70.degree.

This step served to destroy the particle alginate gel-DNA complex and thus release the complexed DNA. This was followed by the addition of 400 .mu.l of a binding buffer (isopropanol / Triton X-100) and a remixing of the particles and an incubation for 2 min. This step served to bind the DNA to the particles. Thereafter, the magnetic particles were separated and the supernatant was discarded. The particles were subsequently washed with alcoholic washing buffers known to the person skilled in the art and finally dried. In the last step, the bound DNA was removed from the particles by adding 50 μl of H ₂ O and transferred to a new reaction vessel.

The 5 ml sample was treated as follows. The sample was transferred to a 15 ml reaction vessel. 150 μl of a 0.5% alginate solution was added to the sample and the batch was mixed briefly. Thereafter, 600 μl of a 1 molar calcium chloride solution and 100 μl of a magnetic particle suspension (MAG Suspension, Analytik Jena AG) were added. The batch was mixed briefly and incubated for 10 minutes. Subsequently, the separation of the magnetic particles by means of a magnet. The supernatant was removed and the magnetic particles were washed with 5 ml of water. After renewed separation of the magnetic particles, the supernatant was completely removed.

400 μl buffer (4 M guanidine thiocyanate, EDTA) and 20 μl proteinase K (20 mg / ml) were added to the complex magnetic particle alginate gel DNA, resuspended and the mixture was transferred with a pipette into a 1.5 ml reaction vessel. This was followed by incubation for 15 min at 70.degree.

This step served to destroy the particle alginate gel-DNA complex and thus release the complexed DNA. This was followed by the addition of 400 .mu.l of a binding buffer (isopropanol / Triton X-100) and a remixing of the particles and an incubation for 2 min. This step served to bind the DNA to the particles. Thereafter, the magnetic particles were separated and the supernatant was discarded. The particles were subsequently washed with alcoholic washing buffers known to the person skilled in the art and finally dried. In the last step, the bound DNA was removed from the particles by adding 50 μl of H ₂ O and transferred to a new reaction vessel.

The detection of the enrichment and subsequent extraction of cell-free DNA was carried out by means of real-time PCR. For this purpose, a human-specific target sequence (estrogen receptor 1) was amplified.

Results real time PCR

Protocol real-time PCR for the amplification of the human-specific target sequence (estrogen receptor 1). Protocol see example 1. sample Ct values 1 ml sample (red curve) 35 / 34.95 5 ml sample (black curve) 32.25 / 32.30

Like the results in 2 show, more DNA is enriched from the 5 ml sample and subsequently isolated than from the 1 ml sample. Thus, the inventive method is also suitable for the enrichment and extraction of cell-free DNA from large-volume samples.

Exemplary Embodiment 3 Enrichment of Genomic DNA from an Aqueous Solution (1 ml) and Direct Release of the Genomic DNA Without Further DNA Extraction. Comparison of the method according to the invention with the method of the patent DE 10 2008 023 297 B4

The initial sample for enrichment was an aqueous solution containing genomic DNA. The enrichment was carried out by means of the method of the patent DE 10 2008 023 297 B4 and by means of the method according to the invention without a centrifugation step. For the method of the patent the commercial product PME free-circulating DNA Extraction Kit (Analytik Jena AG) was used). The process according to the invention was carried out as follows.

30 μl of a 0.5% alginate solution was added to the sample and the batch was briefly mixed. Thereafter, 150 μl of a 1 molar calcium chloride solution and 50 μl of a magnetic particle suspension (MAG Suspension, Analytik Jena AG) were added. The batch was mixed briefly and incubated for 5 minutes. Subsequently, the separation of the magnetic particles by means of a magnet. The supernatant was removed and the magnetic particles were washed with 1 ml of water. After renewed separation of the magnetic particles, the supernatant was completely removed. The complex of magnetic particle alginate gel DNA was subsequently destroyed to release the DNA. For this purpose, 50 .mu.l of a 50 mM sodium citrate solution were added to the particles and the batch was resuspended with a pipette. After a short incubation, the magnetic particles were separated and the supernatant was transferred to a new vessel and subsequently treated with 50 ul of water. The verification of whether the genomic DNA of the sample was enriched without centrifugation was carried out on an agarose gel. As the gel electrophoresis shows, both methods were used to concentrate the genomic DNA from the 1 ml sample. In this case, the process according to the invention is impressive because of its simplicity, since no further centrifugation steps were required.

3 Figure 12 shows the gel electrophoresis on concentrated DNA by the two methods, wherein # 1 represents the 1-DNA ladder, numbers 2 to 5 show the concentrated genomic DNA by the method of the patent DE 10 2008 023 297 and the numbers 6 to 9 depict the concentrated genomic DNA by means of the method according to the invention.

Exemplary Embodiment 4: Enrichment of Viruses and Subsequent Extraction of the Viral Nucleic Acid

The initial sample for the enrichment was human plasma. The plasma sample was added to inactivated yellow fever virus. For the enrichment of the viruses 1 ml sample was used. The 1 ml sample was treated as follows. 30 μl of a 0.5% alginate solution was added to the sample and the batch was briefly mixed. Thereafter, 150 μl of a 1 molar calcium chloride solution and 50 μl of a magnetic particle suspension (MAG suspension, Analytik Jena AG) were added. The batch was mixed briefly and incubated for 10 minutes. Subsequently, the separation of the magnetic particles by means of a magnet. The supernatant was removed and the magnetic particles were washed with 1 ml of water. After renewed separation of the magnetic particles, the supernatant was completely removed.

400 μl buffer (4 M guanidine thiocyanate, EDTA) and 20 μl proteinase K (20 mg / ml) were added to the complex magnetic particle alginate gel DNA and the batch was resuspended with a pipette. This was followed by incubation for 15 min at 60.degree.

This step was to destroy the particle alginate gel-virus complex and thus the release of the complexed viruses and the destruction to release the viral nucleic acid (viral RNA). This was followed by the addition of 400 .mu.l of a binding buffer (guanidine thiocyanate / isopropanol / Triton X-100) and a remixing of the particles and an incubation for 2 min. This step served to bind the viral RNA to the particles. Thereafter, the magnetic particles were separated and the supernatant was discarded. The particles were subsequently washed with alcoholic washing buffers known to the person skilled in the art and finally dried. In the last step, the bound DNA was removed from the particles by adding 50 μl of H ₂ O and transferred to a new reaction vessel.

Reaktionsansatz Pro Probe: YFallF (50 pmol/ µl) 0,1 µl YFallR (50 pmol/ µl) 0,1 µl YFallP (25 pmol/ µl) 0,1 µl dNTP-Mix (12,5 mM) 0,3 µl 10X PCR-Buffer (MgCl₂ included) 1,5 µl Taq-DNA-Polymerase 0,75 U PCR-Grade H₂O add up to 15 µl Amplifikations-/ Hybridisierungskonditionen Schritt 1: Denaturierung 95°C 120 sec Schritt 2 Amplifizierung 45 Zyklen 95°C 4 sec (Messung) 65°C 45 sec Ergebnisse PCR Probe Ct-Werte 1 ml Probe (blaue) 28,8/ 28,9 The detection of the enrichment of the virus particles and the subsequent extraction of the viral RNA was carried out by means of a yellow fever virus-specific real-time PCR. Results real time PCR protocol real time PCR for amplification of the 5 'noncoding region of Yellow Fever virus

reaction Per sample: YFallF (50 pmol / μl) 0.1 μl YFallR (50 pmol / μl) 0.1 μl YFallP (25 pmol / μl) 0.1 μl dNTP mix (12.5 mM) 0.3 μl 10X PCR Buffer (MgCl ₂ included) 1.5 μl Taq DNA polymerase 0.75 U PCR grade H ₂ O add up to 15 μl Amplification / Hybridization Conditions Step 1: denaturation 95 ° C 120 sec step 2 amplification 45 cycles 95 ° C 4 sec (Measurement) 65 ° C 45 sec Results PCR sample Ct values 1 ml sample (blue) 28.8 / 28.9

In 4 it has been shown that viruses can also be enriched and subsequently the viral nucleic acid can be extracted.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102008023297 B4 [0001, 0007, 0019, 0021, 0023, 0035, 0046]
• DE 19856415 C2 [0004]
• DE 102008023297 [0048]

Cited non-patent literature

• Yamamoto et al., Virology 40 (1970) 734; Morandi et al., J. Clin. Microbiol. 36 (1998) 1543-1538 [0004]

Claims (11)

Process for the enrichment of biomolecules and for the removal of biomolecules from a biological sample, characterized in that - in the presence of particles - a biological sample is mixed with an alginate solution and with salts of di- or polyvalent cations or with an acid, wherein forms an alginate gel-biomolecule complex on the particles, which is removed by separation of the particles from the sample and then released from the biomolecules or ingredients of the biomolecules. A method according to claim 1, characterized in that it is magnetic or paramagnetic particles. A method according to claim 1 or 2, characterized in that are used as salts of di- or polyvalent cations calcium, zinc or aluminum salts. Method according to one of claims 1 to 3, characterized in that the biomolecules are cell-free nucleic acids, viruses or subcellular microparticles. Method according to one of claims 1 to 4, characterized in that in the case of magnetic particles, the separation of the particles with the alginate gel-biomolecule complex via a magnet. Method according to one of claims 1 to 5, characterized in that the release of the biomolecules takes place by dissolution of the particles located on the alginate gel-biomolecule complex, wherein the particles and the biomolecules are released. A method according to claim 6, characterized in that the dissolution of the alginate gel-biomolecule complex is carried out by tri-sodium citrate dihydrate or by chelating agents, preferably EDTA. A method according to claim 6 or 7, characterized in that after the dissolution of the alginate gel-biomolecule complex located on the particles, a resuspension of the particles and an incubation takes place and the particles are separated. Method according to one of claims 1 to 8 for the enrichment of nucleic acids and for the removal of nucleic acids from a biological sample, characterized in that the particles on which the alginate gel-nucleic acid complex has formed, after its dissolution simultaneously to the connection of the Complex released nucleic acids serve. A method according to claim 9, characterized in that after the dissolution of the alginate gel-nucleic acid complex and the resuspension of the particles, a binding buffer for binding the nucleic acids to the particles is added and the subsequent isolation of the nucleic acids in a known manner. Kit for carrying out the method according to one of claims 1 to 10, comprising a) an alginate solution b) at least one salt of di- or polyvalent cations or an acid c) particles d) means for separating the particles which have formed a complex of a) and b) with biomolecules e) means for releasing biomolecules bound in a complex of a), b) and c), preferably chelating agents

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