WO2006069413A2

WO2006069413A2 - Method for isolating cells and viruses

Info

Publication number: WO2006069413A2
Application number: PCT/AT2005/000525
Authority: WO
Inventors: Thomas Schlederer
Original assignee: Thomas Schlederer
Priority date: 2004-12-30
Filing date: 2005-12-30
Publication date: 2006-07-06
Also published as: WO2006069413A3; AT501194A1

Abstract

The invention relates to a method for isolating at least one cell, especially a eukaryotic cell and a prokaryotic cell, or a virus, from a complex biological sample in suspension. Said method comprises the following steps: the sample containing the cell or the virus is prepared; the sample in suspension is brought into contact with ferromagnetic particles in such a way that the cell or the virus is joined to the particles; and the particles are magnetically separated from the sample particle suspension.

Description

Method for isolating cells and viruses

The present invention relates to a method for isolating cells and viruses.

The isolation of cells and viruses of various kinds (eg erythrocytes, microorganisms) is carried out in practice in a variety of different methods, the majority of these methods to a separation due to the physical or biological properties of the cells to be isolated, cell fragments (eg microorganisms, microorganism fragments ) or viruses (eg centrifugation, accumulation of microorganisms and viruses in enrichment media or cell cultures). The development of magnetic, especially superparamagnetic, particles which can be used to isolate cells and viruses of any kind from biological samples has led to a simplification of the isolation methods, since the magnetic properties can be used for cells bound to these particles to isolate a liquid or suspension. The particles used in this case are functionalized with specific binding partners which are able to bind structures located on the surface of cells, as a result of which the magnetic particles have a high specificity with respect to the cells to be isolated. Such binding partners may be, for example, antibodies or lectins capable of binding surface epitopes or carbohydrate structures. The development of superparamagnetic particles, which have a high magnetic susceptibility and rapidly lose the magnetic properties as soon as an applied magnetic field is removed, finally led to the economic exploitation of such methods, since such particles themselves acquire magnetic properties when a field is created, whereby a stronger magnetic effect between the individual particles and the magnet can take place.

US 4,910,148 discloses the use of magnetizable particles to isolate cells from a biological sample. In this case, first a sample container is brought into contact with a biological fluid with suspended, magnetizable, functionalized particles and incubated for a defined time. After the incubation to the To isolate cells bound to magnetizable particles, a magnetic field is built up on the container by means of a magnetic device and the supernatant of the biological fluid is removed. Subsequently, the magnetizable particles located on the wall of the container can be further processed. The particles disclosed in this patent comprise a magnetizable material and therefore themselves have no magnetic properties.

US 6 020 210 describes the isolation of biological cell organelles and other biological materials from a biological sample. In this case, a sample containing superparamagnetic particles, on which the material to be isolated is located, is passed over a magnetizable columnar matrix on which the superparamagnetic particles are immobilized after the formation of a magnetic field. After removing the remaining sample-bound liquid from the column, the particles can be eluted from the column matrix after removal of the magnetic field.

The article by Khare et al. (Clin. Microbiol., 42 (2004): 1075-1081) relates to a method for the rapid and sensitive detection of Mycobacterium paratuberculosis in milk and feces. In this case, magnetic particles are added to a sample containing Mycobacterium paratuberculosis, which are due to a corresponding surface functionalization in a position to bind Mycobacterium paratuberculosis. This binding can be accomplished, for example, by the provision of polyclonal anti-Mycobacterium paratuberculosis antibodies. The in Khare et al. However, the magnetic particles disclosed are not ferromagnetic but supermagnetic particles, i. that the particles used in this document can have magnetic properties only in a magnetic field. To carry out further studies, the genomic DNA of the adhering to the magnetic particles mycobacteria is isolated. Following this, the genomic DNA is subjected to a PCR to identify the mycobacteria. Whole cells are not obtained according to this document.

In the article by Grant et al. (Environ. Microbiol. 64 (1998): 3153-3158) discloses a method for isolating Mycobacterium paratuberculosis in milk with the aid of immunomagnetic particles. In this document, DNA particles, which are not ferromagnetic (Dynabeads®), with antibodies directed against Mycobacterium paratuberculosis, which can then be used to bind mycobacteria from a sample to the particles. Also in the work of Djonne et al. (Vet.Microbiol.92 (2003): 135-143) no ferromagnetic particles are used in the detection of Mycobacterium paratuberculosis using immunomagnetic PCR.

WO 2004/053154 A1 relates to a method for the amplification of nucleic acids of a target cell or of a virus which have been isolated from a sample with the aid of magnetic microparticles. In general, the use of magnetic, magnetizable, paramagnetic, ferromagnetic, ferrimagnetic, metal oxide, etc. particles is described, without any particular form being emphasized as having particular effectiveness in the method used. Furthermore, no reference is made to the problem of sample consistency with regard to the analysis result. Finally, with the method described there, no cells or viruses can be obtained in living or propagatable form, since they are destroyed to recover the DNA.

WO 2004/003231 A2 relates to magnetic particles which are capable of binding a substance, these particles consisting of a magnetic and a matrix material and the matrix material having on the surface functional groups which in the liquid phase the Promote disaggregation. The magnetic material may consist of a ferromagnetic or ferromagnetic material. Again, the magnetic particles are used to insulate e.g. DNA or RNA, but also proteins, cells and microorganisms used. The problem of microorganisms present in difficult-to-access sample material is not discussed in this document.

US 5,283,079 relates to a process for producing fluorescent magnetic polymer particles which are suitable for binding molecules such as antibodies or nucleic acids. It is mentioned that the metallic oxide used to produce the particles may have inter alia ferromagnetic properties, but in this case the Purification / separation by centrifugation (instead of magnetic separation) as mandatory, which is why the use of superparamagnetic particles is preferred.

In the US 2004/0023222 Al are particles with a significantly deviating from the norm particle density (7-9 g / cm ³ ) and large (0.5-2 microns) for the selection or isolation of cells, such as bacteria or Viruses, but also of nucleic acids, proteins, hormones and the like disclosed. Although the particles used can also be ferromagnetic, the mixing / separation of the particles takes place exclusively by means of gravity selection ("gravity selection"), which is to be ensured by the significantly increased density of the particles from difficult to access, complex biological sample material is not received.

It is thus an object of the present invention to provide a method by means of which cells, for example eukaryotic cells, such as fungi, in particular yeasts, animal and plant cells, and prokaryotic cells, such as bacteria or other microorganisms, isolated from complex samples and optionally determined or characterized.

Therefore, the present invention relates to a method for isolating at least one cell, in particular a eukaryotic and prokaryotic cell, or a virus from a complex, suspended biological sample comprising the steps:

- Providing the sample containing the cell or the virus

Contacting the sample in suspension with ferromagnetic particles, whereby the cell or virus is bound to the particles, and

- Magnetic separation of the particles from the sample-particle suspension.

Preferably, the cell (s) or virus (s) bound to the ferromagnetic particles are (are) permeated

Contacting the particles with cell- or virus-specific primers, Subjecting the particles of a nucleic acid amplification technique and

qualitatively and / or quantitatively detecting at least one nucleic acid amplification product determines the presence of cells or viruses in the sample.

The term "cell" according to the present invention is understood inter alia microorganisms, eukaryotic and prokaryotic cells, in particular plant and animal cells, bacteria, fungi and the like, whereby cell fragments, such as cell walls, cell wall components (peptidoglycan, S-layers, Lipopolysaccharide, etc.), cell membranes, cell membrane constituents (transmeran protein, etc.) are preferably to be isolated with the method according to the invention are preferably complete (whole, viable and replicable) cells.The method of the invention are preferably pathogenic, especially human and animal pathogenic, Microorganisms, isolated.

"Viruses" include all known viruses for which it is possible to provide binding partners (for example antibodies), the viruses preferably having epitopes or other binding partners accessible on the surface thereof. in particular eukaryotic and prokaryotic cells, the outstanding advantages over the prior art.

Under complex biological samples, which are present in suspension, according to the invention, those samples are understood from which cells are difficult to separate and isolate. Such samples contain, in addition to the aqueous phase, at least one second phase which may be liquid or solid and which has at least 50% worse rheological properties than human blood (eg flowing in a flow test 50% slower than human blood). Surprisingly, in such samples, ferromagnetic particles have proven to be far superior to the superparamagnetic (and of course paramagnetic or ferrimagnetic) particles, especially when removed from the suspension by magnetic separation. Particularly preferred complex biological suspension samples are foodstuffs (with a non-aqueous suspension content of preferably 5-25%, especially 8-20%, eg milk with -15% (predominantly fat) or food grade). troll samples with about 10% non-aqueous suspension content), feces (with preferably 2-20%, in particular 5-10%, non-aqueous suspension content), water filtrates (eg from sewage treatment plants, water supply systems (eg from hospitals) etc.) , Environmental analysis) with a solids content of up to 2.5% (preferably 1-2.5% solids), bottom slurries (preferably 5-20%, especially 8-15% solids), tissue samples (preferably pre-digested samples) with eg 5 -20%, in particular 8-15%, of non-aqueous suspension content and other samples in which the cells to be isolated by paramagnetic particles due to the non-aqueous suspension fraction compared to ferromagnetic particles only to 50%, especially only 30% or even only to 10%, isolate.

Preferably, cells are isolated by the method according to the invention, which occur in complex biological suspensions or tissues.

The "ferromagnetic" property of a particle is characterized by the fact that the elementary magnetic moments of the particles have a parallel order. Ferromagnetic order at room temperature show the metals iron, nickel and cobalt and various alloys thereof. It does not matter whether the metals are crystalline or amorphous. The magnetic order is broken at high temperatures, the ferromagnets then become paramagnetic. The temperature at which the ferromagnetic order disappears is referred to as Curie temperature T _c . At low temperatures, ferromagnetic ordering is also observed for substances that are paramagnetic or superparamagnetic at room temperature, such as the lanthanides gadolinium and dysprosium. Ferromagnetic properties of a substance or particle, which depend on particle size, material and temperature, can be determined, for example, by the inclusion of hysteresis curves (see, for example, abstract book "Scientific and Clinical Applications of Magnetic Carriers", May 20-22, 2400 Lyon , France). This makes it possible in the desired temperature range (eg between 4 and 90 ⁰ C) to determine the particle size and the material of the particles to be used in a simple manner, so that they actually have ferromagnetic properties. It is always essential to the invention that in carrying out the process the particles fer- Thus, for example, the temperature, size of the particles and nature of the particles (for example the exact composition of the particles) are always set so that the particles have ferromagnetic properties in the context of bonding to the magnet. This setting is readily apparent to one of ordinary skill in the art based on his or her expertise and disclosure of the individual method approach described herein.

In Molday RS and MacKenzie D (J Immunol Methods (1982), 52: 353-367), the labeling and isolation of cells with ferromagnetic iron-dextran particles whose core has a size of 15 nm is described. In Molday RS and Molday LL (FEBS Lett (1984), 170: 232-238) red blood cells are labeled by dextran SPIO ("superparamagnetic iron oxide") particles and separated by applying a high gradient field. Such dextran-SPIO particles were prepared by the method of Molday (1982) by Dodd et al. (Biophys J (1999), 76: 103-109) and described as superparamagnetic. The particles with a core size of 15 nm described in Molday (1982) can not be ferromagnetic at a temperature> 273 K.

The sample provided by the method according to the invention can either be used directly in the method or subjected to a corresponding sample preparation. This sample preparation depends on the sample used. For example, a food sample may first be homogenized or suspended. In any case, it is crucial for the sample preparation that the treatment steps have no influence on the later binding behavior of the material to be isolated with optionally functionalized particles.

Surprisingly, it has been found that particularly ferromagnetic particles are particularly well suited for the isolation of cells or viruses from complex biological suspensions. Ferromagnetic particles themselves act magnetically and thus have a high affinity for other magnetically acting materials. Especially in view of the fact that, for example, in US 6,020,210 it is pointed out that ferromagnetic particles with a permanent magnetization are not suitable for the isolation of cells from biological samples, since suspensions with ferromagnetic particles to aggregation, shows the surprising effect of ferromagnetic particles in cell isolation. According to the invention, the ferromagnetic particles may consist entirely or, in a preferred embodiment, only partially of at least one ferromagnetic material, provided that the ferromagnetic properties of the entire particle are retained. Therefore, the cell-particle complexes can be isolated with a magnet from suspensions in a very short time, whereas with superparamagnetic particles, isolation takes a long time. For viscous samples (eg, cheese melts), for example, cell isolation with superparamagnetic particles can not occur in a short time, with many of these particles sticking to the sample matrix itself.

After contacting the sample with the ferromagnetic particles, the resulting suspension for at least 1 minute, at least 5 minutes, at least 10 minutes, is incubated while maintaining the suspension, with the preferred incubation temperature a maximum of 80 ⁰ C, preferably a maximum of 6O ⁰ C, more preferably a maximum of 40 ⁰ C, in particular at most 30 ⁰ C, is. Higher incubation temperatures may be used, for example, for samples which are viscous at room temperature (eg cheese samples), but it has to be taken into account that the higher temperature does not adversely affect or even prevent the binding of the cells to the particles. Nevertheless, room temperature (16 ° to 26 ° C) is preferred as the incubation temperature. The incubation time depends above all on the concentration of the cells or viruses to be isolated in the sample and on the bond strength between these and the optionally functionalized particles. Therefore, the incubation time may vary depending on the samples and particle properties. After the incubation, the particles are separated from the sample particle suspension and optionally subjected to further processing. The separation of the particles is preferably carried out due to the magnetic properties of these particles, since conventional methods, such as filtration, decantation or centrifugation, although in principle are suitable for more complex biological suspensions as samples, however, a significantly worse overall performance of the process - magnetic separation - have. This was too in view of the technical teachings in WO 2004/053154 A, WO 2004/003231 A, US 5,283,079 A and US 2004/0023222 A surprising, in which, however, the problem of isolating cells or viruses on complex biological suspensions as samples were not addressed is. In particular, it has been found that the approach of US 2004/0023222 A (to use particles of higher density) is unsuitable for such complex suspensions, since separation by gravity selection has proved to be ineffective.

After isolation of the cells or of the viruses, these can be analyzed directly on the particle or after the separation thereof by means of a nucleic acid amplification. It has surprisingly been found that the cell / viral particle suspensions can be directly subjected to nucleic acid amplification without the presence of ferromagnetic particles affecting this reaction in any way.

In a preferred embodiment, the complex biological suspension sample is a food sample, fecal sample, biological source, environmental sample, or industrial intermediate or final product.

It has surprisingly been found that although the method according to the invention is suitable for all types of samples, it shows an unprecedented performance especially in the case of very viscous and inhomogeneous samples. For example, the method of the invention can be used to isolate cells or viruses from faeces samples or from cheese melts or cheese suspensions, milk and the like. Industrial processes, such as the production of food, may require regular monitoring of the intermediate and final products for possible contamination. Complex biological suspensions according to the present invention have a non-aqueous suspension content comprising liquid (lipid (fat), multiphase extracts, etc.) and / or solid (biological, organic or inorganic) components. This proportion is usually in the context of the present invention from 1 to 30%, in particular from 3 to 20%, and causes an impairment of the flow or mixing behavior of the suspension, which thus becomes more difficult to analyze than uncomplicated biological suspensions, such as human blood. Of course, the inventive method can also be used to isolate cells and viruses from a sample, for example, to reduce the cell count or the virus titer in the sample. Thus, according to the invention, "isolating" does not just define an analytical or preparative process step.

Preferably, the particles comprise a ferromagnetic material selected from the group consisting of iron, especially magnetite and maghemite, iron alloys, nickel, nickel alloys, cobalt, cobalt alloys, and combinations thereof. Depending on the composition of the material (eg Fe ₇₅ Co _2S over Fe, Fe ₃ O ₄ , the magnetophoretic mobility increases for a given particle diameter.) In order to obtain the ferromagnetic state at the given temperatures, appropriate particle diameters have to be chosen, because even Co- or CoFe nanocrystals have excellent magnetic moments, but are superparamagnetic because of their small size. "A full description of the fabrication, properties, and applications can be found in the abstract book" Scientific and Clinical Applications of Magnetic Carriers "(May 20-22,2004 Lyon, France), in particular the dependence of the ferromagnetic temperature on the particle size, the mobility, depending on size and material, and the hydrodynamic diameter.

According to a further preferred embodiment, the ferromagnetic particles are coated with silicon or silicon compounds, gold, glass, polysaccharides, in particular dextran, plastic, proteins, nucleic acids or combinations thereof. Further materials for the coating or coating process can be found in the abstract book "Scientific and Clinical Applications of Magnetic Carriers" (May 20-22,2004 Lyon, France). A coating which specifically promotes disaggregation, as in WO 2004/003231 A, is superfluous according to the invention and may be disadvantageous in some embodiments, so that the particles according to the invention preferably have no such properties.

A possible functionalization of the ferromagnetic particles presupposes that the surface of these particles can be de-privatized. Ferromagnetic materials can be directly derivatized or functionalized or preferably provided with a corresponding surface, the for example, silicon or silicon compounds, plastic, gold, glass or polysaccharides, such as dextran. In this case, the ferromagnetic material may form the core of the particles or be embedded in a matrix consisting of the above-mentioned coating materials, wherein in such an embodiment, a plurality of ferromagnetic "cores" can be introduced into a particle Finally, functional groups on the surface of the ferromagnetic Bonding particles efficiently binds carboxy-, amine-, tosyl-, epoxy-, hydroxy- and hydrazide groups, streptavidin and His-tag, as already done for paramagnetic and supperparamagnetic particles, but such specific functionalizations, especially With specific binding receptors for certain cells, according to the invention often are not necessary, they are preferably not provided according to the invention.

Preferably, the particles have a diameter of 50 nm to 20 .mu.m, preferably from 200 nm to 5 .mu.m, in particular from 400 nm to 2.5 microns. Preferably, a particle size is chosen which depends on the magnetic material used, so that the magnetophoretic mobility is as high as possible, but the magnetic moment is still small enough, the aggregates formed by moving the suspension, e.g. by pipetting, rotating the Rotamix or in the ultrasonic bath at least partially dissolve.

An important preferred property is that the density of the ferromagnetic particles is preferably not higher than 5 g / cm ³ (eg, 0.5-5 g / cm ³ ). For practical reasons, a particularly preferred range of density is from 1.5 to 4 g / cm ³ , in particular from 1.75 to 2.75 g / cm ³ . The ferromagnetic properties are optimally balanced against the greatest possible similarity of the densities of the cells to be isolated.

In this context, "diameter" is the greatest distance between the points of intersection of a body with a straight line passing through the body center (body center).

The ferromagnetic particles may be functionalized, in particular with at least one cell- or virus-specific binding partner, but this embodiment is just be the Serial testing not preferred.

The binding partners provided on the particles have a specificity with respect to the cells or viruses to be isolated. In any case, it must be ensured that the binding partner specifically recognizes those parts that are actually accessible in the sample. For example, ligands or epitopes (for example carbohydrate structures, antigens) located on the cell surface are suitable for this purpose.

The at least one binding partner may be e.g. an antibody or antibody fragment, protein A, protein G, a lectin, a peptide, a nucleic acid or a combination thereof.

Nevertheless, it is quite possible to isolate cells, which themselves have magnetic properties, from a sample directly with ferromagnetic beads, without these having to be functionalized. Such cells include microorganisms such as e.g. Magnetospirillum magneticum or Aquaspirillum magnetotacticum.

According to the particularly preferred embodiment, the separation can be effected by means of a magnetic device.

Although the separation of the particles from the sample-particle suspension can, as mentioned, be achieved by methods such as centrifugation, the application of a magnetic field by means of a magnetic device is for the isolation of cells or viruses from complex biological Suspensions clearly preferred. In this case, a magnetic field is applied to the container at the desired time, whereby the ferromagnetic particles are immobilized on the wall of the vessel. Subsequently, the supernatant can be easily removed.

Preferably, the magnetic device is designed as a magnet, in particular as an electric or permanent magnet.

The use of an electromagnet for separating off the particles has the particular advantage that the strength of the magnetic field can be influenced by the application of a current flow. This makes it possible to attach the electromagnet permanently to a device (eg container, hose) and to control the magnetic field as needed. In contrast, the magnetic field is completely independent of the presence of a current flow, which, however, leads to the variation of the magnetic field. field strength of the magnet itself must be removed. For example, electromagnets, for machines with high sample throughput permanent magnets, can be used for individual samples.

The method according to the invention can also be used to first isolate cells or viruses from a sample in order subsequently to detect them in a detection step and, if appropriate, to determine their concentration in the sample. Again, the sample is first brought into contact with functionalized ferromagnetic particles. This makes it possible that the thus insulating cells or viruses can be immobilized on these particles. Subsequent to this binding step, the particles are separated from the sample particle suspension, wherein a separation can be carried out by methods such as centrifugation or filtration or by the application of a magnetic field. Finally, in order to qualitatively and quantitatively determine the existing cells or viruses, the particles on which the cells or viruses or fragments thereof are located are mixed with corresponding specific primers. After performing a nucleic acid amplification, the presence of cells or viruses in the sample is optionally qualitatively and / or quantitatively determined by the detection of at least one amplification product. One (or more) fragment specific for a cell or virus produced by the nucleic acid amplification technique may be prepared by known methods, such as e.g. Southern blot, gel electrophoresis (with silver or Coomassie staining), photometry.

The cell- or virus-specific primers to be used in this method can be derived, for example, from already known nucleic acid data.

Preferably, between the separation of the particles and the contacting of the particles with cell- or virus-specific primers, the cells bound to the particles will be at least partially lysed.

By lysing the cells bound to the particles, the nucleic acids present in these cells (including DNA, RNA, mRNA) are made accessible to the primers. The lysis of the cells is carried out by methods known in the art, in particular by a heat shock (eg 95 ⁰ C) or by chemical or biochemical reagents, such as lysozyme. In order to inhibitors or enzymes, in particular proteases and restriction enzymes, which could interfere with nucleic acid amplification, the particles can be treated after separation with appropriate solutions (solutions comprising guanidine thiocyanate, for example).

Preferably, the nucleic acid amplification is a chain reaction technique (PCR technique), which is selected from the group consisting of real-time PCR, in particular, TaqMan PCR (labeled with SYBR Green primer / probes), nested PCR ( "nested PCR ^ΛΛ), inverse PCR, Multiplex PCR, asymmetric PCR, reverse transcriptase PCR and combinations thereof.

In principle, all nucleic acid amplification techniques known to the person skilled in the art can be used according to the invention. However, should a quantitative statement about cells or viruses be made in the samples, real-time PCR techniques (e.g., TaqMan PCR) are particularly well-suited. With such techniques, a statement can already be made in the course of the PCR as to whether and in which concentration a specific fragment which is detected by the attachment of a fluorescent probe is present in the sample.

With the method according to the invention, preferably pathogenic cells, in particular pathogenic microorganisms, such as e.g. Mycobacteria, and viruses are determined in a sample.

However, the central aspect of the present invention relates to the use of at least partially ferromagnetic particles, which are optionally functionalized, for the isolation of cells or viruses for further cultivation or propagation.

Another aspect of the present invention relates to the use of ferromagnetic particles, optionally functionalized, for the combined isolation and detection of cells or viruses.

Another aspect of the present invention relates to a kit for the combined isolation and determination of cells or viruses in a sample comprising

functionalized or functionalizable ferromagnetic particles and

- Cell or virus specific primers.

A kit according to the invention may comprise both functionalized and functionalizable ferromagnetic particles. By providing functionalizable ferromagnetic particles, the user of this kit can apply any binding partners to the particles. For example, the particles to be functionalized have epoxy groups on the surface which can react directly with amino groups of an antibody so as to bind it to the particle.

Another aspect of the present invention relates to a method for the isolation and detection of Mycobacterium paratuberculosis, in particular from feces samples, by the use of a method according to the present invention.

Initially, if appropriate, functionalized ferromagnetic particles capable of binding with M. paratuberculosis are brought into contact with a sample and isolated. The isolated cell-particle complex is then spiked with specific primers directly or after lysing the cells on the particles (e.g., P.H. Vary, Journal of Clinical Microbiology (1990) 933-937). Preferably, the following primers are used: forward primer: MP139U24 5'-CCGCTAATTGAGAGATGCGATTGG-3 '; reverse primer: MP221L22 5'-GTGCCACAACCACCTCCGTAAC-3 ', MP219L24: 5'-GTGCCACAACCACCTCCGTAACCG-S'; Probe for real-time PCR: MP204L17: 5'-CGTCATTGTCCAGATCA-S ', 5'-TCATTGTCCAGATCA-3'.

When the isolation of the cells, especially the mycobacteria (e.g., Mycobacterium tuberculosis), is used to provide these cells, they may preferably be introduced into the appropriate medium immediately with the particles for cultivation, and preferably cultured with the particles. As a result, extremely pure cultures of mycobacteria are provided, which is particularly important in view of the long culturing times of these microorganisms (up to 18 months) and the lack of a selective nutrient medium.

The invention will be further described by way of example by the following figures and the following example, but without being limited thereto.

FIG. 1 shows an agarose gel on which 10 μl PCR amplicon of PCRs were reacted with the particles resulting from point 4 (example) (only stored 040813-2, activated 040813-1 and coated 040811-1).

Figure 2 shows an agarose gel: lOμl of the amplicon are on Analyzed gel, with a specific band seen in both approaches: lane 1 is isolation as in point 4, lane 3 as described in point 5; for lane 2 and lane 4, the particles were washed as in items 4 and 5, but the starting milk was diluted 1:10 with cell lysis buffer.

FIG. 3 shows an amplification plot of a real-time PCR with SybrGreen (point 11, example).

Example: Isolation, detection and quantification of Mycobacterium paratuberculosis

1. General reagents

TPBS: 8g NaCl (Sigma, # S9625), 1, 15g Na ₂ HPO ₄ x 2H ₂ O (Merck, # 106580), 0.2g KCl (Merck, # 104936), 0.2g KH ₂ PO ₄ (Merck, # 104873), 100 μl Tween20 (Merck, # 822184) were made up to 11 in water.

MES buffer: Set 9.76g MES (Sigma, # M3023) to pH = 5.5 with ION NaOH and make up to 11.

MOPS Buffer: Set 5.23g MOPS (Sigma, # M1254) to pH = 7.5 with ION NaOH and make up to 11.

Denaturation buffer: 56 mM NaOH in water.

Hybridization Buffer: O, 1N NaH ₂ PO ₄ .H ₂ O in water.

CT wash buffer: 20 mM Tris, 15 mM NaCl in water pH = 7.4.

Inhibitor Removal Buffer: 2.5M guanidine thiocyanate, 10 mM Tris, ImM EDTA in water, pH = 7.0 with ethanol to 70% ethanol.

Cell lysis buffer: 8g NaCl, 1.15 g Na2HPO4x2H2O, 0.2 g KCl, 0.2 g KH2PO4, lOOμl Tween20, 0,508g MgCl ₂ × 6H ₂ O, 0,788g TrisHCl, 0,605g Tris base, 5 ml Triton X-IOO, 54 , 75 sucrose are made up to 500 ml with dH ₂ O.

2. ferromagnetic particles

SIMAG-MP / Carboxyl (Chemicell, Berlin), aqueous dispersion of magnetic silicate, core is magnetite, matrix is non-porous silicate, size lμm, surface area ~ 25m ² / g, -1.5 x 10 ⁸ particles / g, density ~ 2.25 g / cm ³ , magnetization is ferromagnetic, carboxyl is functional group with 12 atoms spacer, degree of carboxylation is 350 μmol COOH / g, storage buffer is ddH ₂ O.

2.1. Particle storage

0.8 ml of ferromagnetic particles (ChemiCell, SIMAG-MP / TCL) suspension are deposited on the magnet (MagnetOn 4T, Aureon Biosystems, # M010104), washed with 4 ml of StabilZyme AP (Surmodics, SAOL-100), taken up in 20 ml of StabilZyme ( Final concentration of 10mg particles / mL) and stored in the refrigerator. 2.2. Particle activation

0.8 ml ferromagnetic particles (ChemiCell, SIMAG-MP / TCL) suspension are deposited on the magnet (MagnetOn 4T, Aureon Biosystems, # M010104), resuspended in ImI MES, the supernatant removed from the magnet and incubated with 25mg carbodiimide (Sigma, # E7750 ) in 1 ml of MES buffer for 15 minutes on rotamix (RMl at 15 rpm). The particles are washed with 1 ml of MOPS buffer and rotated with 900 μl of MOPS buffer for 2 hours at RMI. Afterwards 4ml StabilZyme AP (Surmodics, SAOl-100) are added and turned overnight at the RMl. By depositing the particles on the magnet, resuspending and reprecipitating, the particles are washed 2x with ImI TPBS and Ix with ImI StabilZyme. Finally, the particles are taken up in 20 ml of StabilZyme (final concentration of 10 mg of particles / mL) and stored in the refrigerator.

2.3. Antibody coating

0.8 ml ferromagnetic particles (ChemiCell, SIMAG-MP / TCL) suspension are deposited on the magnet (MagnetOn 4T, Aureon Biosystems, # M010104), resuspended in ImI MES, the supernatant removed from the magnet and incubated with 25mg carbodiimide (Sigma, # E7750 ) in 1 ml of MES buffer for 15 minutes on Rotamix (RMl at 15rρm) activated. The particles are washed with ImI MOPS buffer and spiked for 2 hours at RMI with 900 μl of antibody in MOPS buffer (QED Bioscience, 18101-18104). Afterwards 4ml StabilZyme AP (Surmodics, SAOl-100) are added and turned overnight at the RMl. By depositing the particles on the magnet, resuspending and reprecipitating, the particles are washed 2x with ImI TPBS and Ix with ImI StabilZyme. Finally, the particles are taken up in 20 ml of StabilZyme (final concentration of 10 mg of particles / mL) and stored in the refrigerator.

3. Preparation of the microplates for Hybridisierunqsassay streptavidin Roche is diluted to a concentration of 2μg / ml in carbonate coating buffer and coated the wells of white MaxiSorp microplates (Nunc, Style No. 437-591) overnight at 4 ⁰ C with lOOμl , The microplates are washed 4x with TPBS and lOOμl of the sample bio-TCATTGTCCAGATCA in TPBS (30 pmol/mL) bound overnight at 4 ^0C. After 4x washing with TPBS, the plates are dried and sealed.

4. Isolation of M. paratuberculosis from milk

ImI of a suspension of M. paratuberculosis in raw milk (Andiatec, Stuttgart) is mixed with 10 μl of a particle suspension and incubated for 30 minutes with occasional shaking. Thereafter, the particles are deposited on the magnet and washed 4 times with CT wash solution by respective deposition on the magnet. The particle-bacteria complex is now available for further analysis.

5. Isolation of M. paratuberculosis DNA from milk by inhibitor removal buffer

In a suspension of M. paratuberculosis in raw milk (Andiatec, Stuttgart), 10 μl of the particle suspension are added and the mixture is incubated for 30 minutes with occasional shaking. Thereafter, the particles are deposited on the magnet and washed twice with Inhibitor Removal Buffer, once with CT Wash Solution / 70% EtOH, and once with CT Wash Solution by respective deposition on the magnet. The particle-DNA complex is now available for further analysis.

6. Preparation of M. paratuberculosis DNA from milk ImI of a suspension of M. paratuberculosis in raw milk

(Andiatec, Stuttgart) is mixed with 10 μl of the particle suspension and incubated for 30 minutes with occasional shaking. Thereafter, the particles are deposited on the magnet and washed 4 times with CT wash solution by respective deposition on the magnet. The particle-bacteria complex is distilled in 50 μl. Water resuspended, 10 min. heated at 95 ° C, then the particles deposited on the magnet and transferred the supernatant into a fresh tube.

7. Preparation of high purity M. paratuberculosis DNA ImI of a suspension of M. paratuberculosis in raw milk

(Andiatec, Stuttgart) is mixed with 10 μl of the particle suspension and incubated for 30 minutes with occasional shaking. Thereafter, the particles are deposited on the magnet and washed twice with Inhibitor Removal Buffer, once with CT Wash Solution / 70% EtOH, and once with CT Wash Solution by respective deposition on the magnet. The particle-DNA complex is distilled in 50 μl. Water resuspended, 10 min. heated at 95 ° C, then the particles deposited on the magnet and transfer the supernatant into a fresh tube.

8. PCR with particles

The particles resulting from point 4 (only stored 040813-2, activated 040813-1 and coated 040811-1) are mixed with 50 μl PCR amplification mix (Andiatec, Stuttgart) and It. Manufacturer's information amplified. 10μl of the amplicon is analyzed on the gel showing a specific band in all three batches (Figure 1).

9. PCR with bacterial particle complex and bacterial DNA complex

The particles resulting from point 4 or point 5 were mixed with 50 μl of PCR amplification mix (Andiatec, Stuttgart) and amplified according to the manufacturer's instructions. 10μl of the amplicon is analyzed on the gel, with a specific band seen in both batches. Lane 1 is insulation as in point 4, lane 3 as described in point 5. For lane 2 and lane 4, the particles were washed as in items 4 and 5, however, the starting milk was diluted 1:10 with cell lysis buffer (Figure 2).

10. Asymmetric PCR with DNA

The forward primer was taken directly from the literature (PH Vary, Journal of Clinical Microbiology (1990) 933-937). This primer is designated MP139U24 (5'-CCGCTAATTGAGAGATGCGATTGG-3 '). However, because the primer pair described in this document did not have sufficient specificity (due to Clustal X (1.81) Multiple Sequence Alignment analyzes), the reverse primer was placed elsewhere. This has the advantage that the amplification product was very short (104bp), at 95 ⁰ C for a second are denatured and thus had normal as PCR Real-time very quickly and was efficient. In addition, a two-step PCR can be carried out because the primers had a high melting temperature.

Following the reverse primer, the sample was designed to exploit the cooperative effect of hybridized DNA if all base pairs match. This accumulation of differences in the sequences of the capture probe plus the sequence-specific primer provides for very good PCR specificity.

It has been found that the reverse primers MP221L22 (5 ^I -GTGCCACAACCACCTCCGTAAC-3 ^• ) and MP219L24 (5'-GTGCCACAACCACCTCCGTAACCG-3 ') together with the above-mentioned forward primer and the probes MP204L17 (5'-CGTCATTGTCCAGATCA) and 5 '-TCATTGTC- CAGATCA showed the best results. 1 μl of a dilution series of the DNA solution isolated from point 6 was labeled with 4 pmol / 20 μl with the fluorescein labeled primer MP139U24: 5'-F-CCGCTAATTGAGAGATGCGATTGG-S 'and 2 pmol / 20 μl It amplifies the primer MP219L24: 5'-GTGCCACAACCACCTCCGTAACCG-S 'in Ix SybrGreen Supermix from BioRad on the Biometra TGradient cycle in a total volume of 20 μl asymmetrically.

PCR parameters:

5 μl of the amplicons were incubated either with 10 μl of denaturation buffer for 10 min and applied to the plate with 95 μl of hybridization buffer. Alternatively, 5 μl amplicon with 95 μl TPBS are applied to the microplate. Incubate with shaking for 15 min at room temperature, wash 4x with TPBS, incubate 100 μl of anti-fluorescein-AP (Roche) 1: 10,000 in StableZyme at room temperature with shaking, wash 4x with TPBS, incubate 100 μl LumiPhos Plus (Lumigen) for 15 min at room temperature and on the luminometer (Mediators PhL) with 1 sec integration time.

11. Real-time PCR with SvbrGreen 'and DNA solution 1 μl of dilutions of the DNA solution isolated from point 6 was incubated with 4 pmol / 20 μl with the primer MP139U24: 5'-CCGCTAATT-GAGAGATGCGATTGG-3' and 4 pmol / 20 μl with the primer MP219L24: 5 '-GTGCCACAÄCCACCTCCGTAACCG-3' amplified in Ix SybrGreen Supermix from BioRad on iCycle in 20μl total volume.

PCR parameters (results in FIG. 3): Cycle 1 repeats 1 3 min 95 ⁰ C A3 Negative control

Cycle 2 repetitions 50 1 sec 95 ⁰ C A4 Negative control

30 sec 67 ⁰ C A5 10 ^"10 dilution

Cycle 3 repeats 1 - 25 ⁰ C Aβ 10 ^"9 dilution

12. Real-time PCR with Svbr Green with and without particles lμl of the 10 ^{~ 7} dilution of point 6 isolated DNA solution was incubated with 4 pmol/20μl with the primer MP139U24: 5'-CCGCTAATT-GAGAGATGCGATTGG-3 'and 4 pmol/20μl with the Primer MP219L24: 5'-GTGCCACAACCACCTCCGTAACCG-3 'amplified in Ix SybrGreen Supermix and 100μg ferromagnetic particles on iCycle with 20μl total volume. The particles were previously incubated with milk and washed as indicated under items 4 and 5.

PCR parameters:

Results (Threshold Cycle - "Threshold Cycle")

Without particles Addition With particles additive

Point 4 26.4 26.7

Point 5 25.7 26.1

13. Real-time PCR with SvbrGreen and DNA Solution 1 μl of a dilution series of the DNA solution isolated from point 6 was mixed with 4 pmol/20 μl MP139U24: δ'-CCGCTAATTGAGAGATGCGATTGG-3 'and 4 pmol/20 μl with the primer MP219L24:

5'-GTGCCACAACCACCTCCGTAACCG-S 'in Ix SybrGreen Supermix from Bio-Rad on the iCycle with 20μl total volume amplified and analyzed real-time.

To demonstrate the robustness, sperm DNA was added to the sample.

PCR parameters:

Results (threshold cycle threshold cycle):

Without DNA Addition With 200ng of herring sperm DNA

_lo -io dilution 34.4 40, 8

₁₀ - ₉ dilution 31.9 35, 8

10 " ⁸ dilution 28.5 32, 2 io- ⁷ dilution 25.5 29, 2

The melting curve showed a melting temperature of 79 ⁰ C for the product of the internal control and 86 ° C for the product of M. paratuberculosis PCR.

14. Comparison of the Speed of Fan Fens Ferromagnetic Particles with Superparamagnetic Particles

20 μg of alkaline phosphatase coated particles (streptavidin particles with excess biotin-AP incubated and washed with TPBS) were suspended in 1 ml TPBS in 1.5 ml Eppendorf cups and incubated for 2 seconds with the Aureon Separation Pen (M020101, Aureon, Vienna) recovered from the suspension and then resuspended in ImI TPBS. 10 μl of the starting suspension and of the isolated beads were measured in a white microplate after 15 minutes of incubation with 100 μl LumiPhos Plus (Lumigen, USA) on the mediators PhL with 1 second integration time.

15. Discussion

Contrary to the reservations from the literature, ferromagnetic particles were used for the isolation of cells and, surprisingly, it was found that such particles are very well suited for cell isolation. This is of great importance because only ferromagnetic particles can be deposited in an acceptable time from difficult sample material, such as raw milk, feces or high solids samples, with a magnet from the sample suspension to the vessel wall.

In the example ferromagnetic particles were used, which can nonspecifically bind bacteria, as well as ferromagnetic particles, which are functionalized with an antibody. Furthermore, it could be shown that the particle-cell complex can be used directly in the PCR. In order to remove substances which could adversely affect the PCR or distort the results, an inhibitor removal buffer was used and it was shown that in this case the DNA remains bound to the particles and the DNA-particle complex is used in the PCR can be.

To simplify the evaluation of the PCR, it has been shown that the product can be hybridized to an asymmetric PCR without denaturation.

Real-time PCR is very important for quantification today, and it has been shown that ferromagnetic particles can be used in such applications.

Furthermore, it was possible to develop a highly sensitive and highly specific PCR for the detection of M. paratuberculosis DNA and to show that even after 80 runs no primer dimers appear. The specificity was further increased with the same primers in a touch-down PCR.

Claims

claims:

A method for isolating at least one cell, in particular a eukaryotic and prokaryotic cell, or a virus from a complex, suspended biological sample comprising the steps:

- Providing the sample containing the cell or the virus

- Magnetic separation of the particles from the sample-particle suspension.

2. The method according to claim 1, characterized in that the bound on the ferromagnetic particles (s) cell or virus (s) by

Contacting the particles with cell or virus specific primers,

Subjecting the particles of a nucleic acid amplification technique and

qualitatively and / or quantitatively detecting at least one nucleic acid amplification product the presence of cells or viruses in the sample is determined.

A method according to claim 1 or 2, characterized in that the sample is a food sample, feces sample, biological origin sample, environmental sample or industrial intermediate or final product.

4. The method according to any one of claims 1 to 3, characterized in that the particles comprise a ferromagnetic material selected from the group consisting of iron, in particular magnetite and maghemite, iron alloys, nickel, nickel alloys, cobalt, cobalt alloys and combinations thereof.

5. The method according to any one of claims 1 to 4, characterized in that the ferromagnetic particles are coated with silicon or silicon compounds, gold, glass, polysaccharides, in particular dextran, plastic or combinations thereof.

6. The method according to any one of claims 1 to 5, characterized in that the particles have a diameter of 50 nm to 20 .mu.m, preferably from 200 nm to 5 .mu.m, in particular from 400 nm to 2.5 .mu.m.

7. The method according to any one of claims 1 to 6, characterized in that the ferromagnetic particles are functionalized.

8. The method according to claim 7, characterized in that the ferromagnetic particles are functionalized with at least one cell or virus-specific binding partner.

9. The method according to claim 7 or 8, characterized in that the binding partner is an antibody or antibody fragment, protein A, protein G, a lectin, a peptide, a nucleic acid or a combination thereof.

10. The method according to any one of claims 1 to 9, characterized in that the separation takes place by means of a magnetic device.

11. The method according to claim 10, characterized in that the magnetic device is designed as a magnet, in particular as an electric or permanent magnet.

12. The method according to any one of claims 2 to 11, characterized in that between the separation of the particles and the contacting of the particles with cell-or virus-specific primers, the cells or viruses bound to the particles are at least partially lysed.

13. The method according to any one of claims 2 to 12, characterized in that the nucleic acid amplification technique is a polymerase chain reaction technique (PCR technique).

14. The method according to claim 13, characterized in that the polymerase chain reaction technique (PCR technique), selected from the group consisting of real-time PCR, in particular TaqMan PCR, nested PCR, inverse PCR, multiplex PCR, asymmetric PCR, reverse transcriptase PCR and combinations thereof.

15. The method according to any one of claims 1 to 14, characterized in that the cells selected from the group consisting of mycobacteria, Listeria, Salmonella, Campylobacter, and combinations thereof, are.

16. The method according to any one of claims 1 to 15, characterized in that the viruses are selected from the group consisting of enteroviruses, CMV, hepatitis, HIV and combinations thereof.

17. Use of at least partially ferromagnetic, optionally functionalized, particles for the isolation of cells or viruses.

18. Use of at least partially ferromagnetic, optionally functionalized, particles for the combined isolation and determination of cells or viruses.

19. Kit for combined isolation and determination of cells or viruses in a sample comprising

ferromagnetic, optionally functionalized or functionalizable particles and

- Cell or virus specific primers.