EP1570077A1

EP1570077A1 - Method for purifying nucleic acids

Info

Publication number: EP1570077A1
Application number: EP03779999A
Authority: EP
Inventors: Uwe Michelsen; Willi Roth; Kai Hourfar
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2002-12-13
Filing date: 2003-11-20
Publication date: 2005-09-07
Also published as: WO2004055207A1; AU2003288124A1; DE10258258A1

Abstract

The invention relates to a method for isolating nucleic acids from biological, in particular, large-volume samples while using an inorganic supporting material. The binding of nucleic acids to the supporting material ensues under acid conditions, and the elution ensues under basic conditions. The invention results in a large increase in the yield of isolated nucleic acids by virtue of the fact that a proteinase is added to the washing buffer.

Description

Process for the purification of nucleic acids

The invention relates to a process for the purification or isolation of nucleic acids, in particular from large-volume liquid samples using inorganic oxidic carrier materials in combination with suitable buffer systems for acidic binding and basic elution of the nucleic acids. The effectiveness of the purification could be increased considerably compared to known processes by adding a proteinase to the washing buffer.

The isolation and purification of nucleic acids from complex biological starting materials, such as body fluids, is of great importance e.g. for the detection of viral diseases.

Classic methods for isolating nucleic acids from complex starting materials such as blood or plasma begin with the lysis of the biological material using a detergent and / or a chaotropic salt. These substances enable the inactivation and denaturation of proteins and enzymes. In the following, organic solvents such as e.g. Phenol and / or chloroform, ethanol precipitation and centrifugation steps cleaned the nucleic acids. Not only are these methods very labor intensive and time consuming, they are also difficult to automate.

More modern methods are based on the use of solid phases or carrier materials. No. 5,234,809 describes a method in which the nucleic acids are bound to a silica solid phase in the presence of chaotropic agents such as guanidinium salts. WO 99/40098 discloses a method in which the nucleic acids are bound to silica solid phases under acidic buffer conditions. However, there is still a need not only to isolate nucleic acids quickly and easily but also quantitatively, in particular from large sample volumes. Particularly effective methods are required above all if the sample is of biological origin and contains very few of the nucleic acids to be detected. An example of this is the detection of viral nucleic acids in body fluids, such as plasma or blood.

For the detection of the isolated viral nucleic acids e.g. known nucleic acid amplification techniques, e.g. Polymerase chain reaction (PCR), branched DNA detection (bDNA) or nucleic acid sequence based amplification (NASBA) are used.

Prerequisite for successful and sensitive detection of viral particles in body fluids, e.g. Hepatitis B or C virus, (HBV, HCV) or human immunodeficiency virus (HIV), using the nucleic acid amplification techniques mentioned is the efficient, quantitative isolation of the viral nucleic acids (DNA or RNA) in the purest and most native form possible. The detection limit of the commercially available test systems is approximately 100 to 400 nucleic acid copies per ml of plasma. Most test systems are also only for one virus type and are designed for the use of relatively small sample volumes (100-500 l plasma).

This results in e.g. For two important fields of application, there is still a problem due to the sensitivity of the detection systems being too low: 1.) Blood banks, which form so-called mini pools due to high sample volume and cost pressure (from up to 100 individual donor samples), must potentially come from a single serum Can detect viruses in the pool.

2.) The therapy of HCV and AIDS patients requires the highly sensitive monitoring of the virus concentration in the blood, since these viruses are very low Number of copies (<10 copies / ml) can persist despite therapy and their detection is crucial for the continuation of therapy.

Attempts to increase the sample volume mostly fail due to the limited binding capacity and / or the binding specificity of the solid phases used.

As an alternative, preconcentration methods are currently used to achieve high sensitivity. Cells or viruses from a sample are enriched by filtration, ultra-high centrifugation or by affinity binding to an antibody-solid phase complex from the sample. After this concentration step, the DNA or RNA is extracted and suitably purified for the detection reaction. These known concentration processes are not only labor-intensive and costly, they are also not automated and also involve the potential risk of losses, cross-contamination and sample mix-ups.

It was therefore an object of the present invention to provide a process for the purification of nucleic acids, in particular from large-volume biological samples, which does not require a preconcentration process and which exceeds the methods known hitherto, in particular in terms of the sensitivity and effectiveness of the isolation.

It has been found that the method for isolating nucleic acids known from WO 99/40098 by binding to solid support materials under acidic buffer conditions can be considerably improved by adding a proteinase to the washing buffer. The method according to the invention is particularly suitable for isolating nucleic acids from large-volume samples. The present invention therefore relates to a method for isolating nucleic acids from liquid samples, characterized by the following method steps: a) providing a liquid sample which contains nucleic acids; b) providing a carrier material made of an inorganic hydroxyl-containing oxidic material; c) mixing the sample from step a) with a binding buffer so that the pH of the mixture is below 7; d) treating the sample from step c) acidified by means of a binding buffer with the carrier material from step b), the nucleic acids being bound to the carrier material; e) separating the carrier material with the bound nucleic acids from the rest of the sample and the binding buffer; f) washing the carrier material with a washing buffer which contains at least one component which degrades proteins; g) Elution of the nucleic acids bound in step d) from the support material with an alkaline elution buffer.

In a preferred embodiment, the liquid sample provided in step a) consists of cell-free body fluid.

In a preferred embodiment, the binding buffer additionally contains at least one lysing component, such as a non-ionic detergent.

In a preferred embodiment, the binding buffer additionally contains at least one sulfate salt, e.g. Ammonium sulfate, potassium sulfate, cobalt sulfate or zinc sulfate.

In a further preferred embodiment, the liquid sample provided in step a) has a volume of over 2 ml. In a further preferred embodiment, the sample provided in step a) consists of a mixture of at least 10 individual samples. Typically, aliquots are taken from each individual sample and mixed. In the same way, the individual samples can be completely mixed and the sample to be provided in step a) can be taken from this mixture. The procedure of mixing several individual samples, ie the formation of sample pools, is particularly advantageous for applications in blood banks, since it offers the possibility of automatically mixing and testing, for example, usually 10 to 100 individual samples or aliquots of the individual samples before nucleic acid isolation ,

In a preferred embodiment, the volume of the elution buffer in step g) is less than 1/10 of the volume of the sample provided in step a).

In a preferred embodiment, one or more balls are added in step f) and or step g) to support the resuspension of the carrier material.

The present invention also relates to a test kit for carrying out the method according to the invention at least comprising a carrier material made from an inorganic hydroxyl-containing oxidic material, a binding buffer and a washing buffer which contains at least one component which degrades proteins.

The present invention also relates to the use of the method according to the invention for the isolation and detection of viral nucleic acids from body fluids. The method according to the invention is so sensitive that samples can be used which contain viruses in a copy number of less than 200 per ml sample, particularly preferably in a copy number of less than 50 per ml sample. Further information on Figures 1 to 4 can be found in Examples 3 to 6.

Liquid samples in the sense of the present invention are e.g. Buffer solutions and homogenates or complex biological fluids, preferably samples of biological origin, in particular human and animal body fluids such as blood, plasma, serum, urine, feces and liquor. Cell-free body fluids such as plasma, serum, cerebrospinal fluid or urine are particularly preferred.

The method according to the invention is particularly well suited for the isolation of nucleic acids from large-volume samples, i.e. not only from samples between 50 μl and 1 to 2 ml, but in particular from samples with a volume of more than 2 ml, especially from samples with a volume between 2 and 10 ml.

According to the invention, a material which carries hydroxyl groups on the surface, in particular inorganic oxidic materials, can be used as the solid phase or carrier material. Suitable materials are, for example, silica gel, silicates, metal oxides such as iron hydroxides, hydroxyapatite or glass. Materials which have Si-OH groups on the surface are preferred. The carrier materials can be porous or non-porous, in the form of, for example, particles, fibers, membranes, filters or appropriately modified vessel walls. Particulate materials are generally preferred because they have greater binding capacity and kinetics, particularly in larger sample volumes. Magnetizable particles are particularly preferred since their separation from a suspension is easy to automate. A according to the invention particularly suitable solid phase is magnetizable silica particles such as silica particles MagPrep ^® (Merck KGaA, Darmstadt). One of the binding buffers mentioned below is preferably used as the equilibration buffer for the carrier material.

According to the invention, components that break down proteins are, in particular, enzymes such as proteinases, particularly preferably proteinase K.

According to the invention, nucleic acids are ribonucleic acids (RNA) or deoxyribonucleic acids (DNA), such as e.g. genomic DNA or RNA, especially viral genomic DNA and / or RNA.

The nucleic acids from the sample are bound to the carrier material by simply lowering the pH to below pH 7, preferably below pH 6. For this purpose, a binding buffer is used which has a pH range from 1 to 6, preferably from 3 to 5. can maintain. Suitable buffers are e.g. Formate, acetate, citrate buffers or other buffer systems that have sufficient buffer capacity in the pH range mentioned.

The buffer concentration is selected depending on the volume and buffer capacity of the sample liquid to be examined. Preferably a binding buffer with a concentration of 100-200 mM is used which has a pH between 4 and 5, e.g. a buffer of acetic acid, which was adjusted to a pH between 4 and 5 with sodium hydroxide solution, potassium hydroxide solution or with Tris base. Nuclease inhibitors can be added to the binding buffer; suitable nuclease inhibitors are known to the person skilled in the art.

A binding buffer according to the present invention contains neither ionic detergents nor other ions in high concentrations, so that there are no chaotropic conditions during the binding of the nucleic acids to the carrier material. The binding buffer preferably contains no ionic detergents or chaotropic substances. If such connections are nevertheless contained, then the Concentration of the ionic detergents and chaotropic substances is selected such that the mixture of sample and binding buffer contains neither ionic detergents nor chaotropic substances in concentrations> 500 mM, preferably> 200 mM. For this purpose, the binding buffer particularly preferably contains neither ionic detergents nor chaotropic substances in concentrations> 500 mM, preferably> 200 mM.

In a preferred embodiment, the binding buffer additionally contains at least one lysing component, such as, for example, nonionic detergents. NP are preferred as the lysing component Triton ^®, Tween ^®, used 40 or mixtures thereof. The addition of the lysing component is particularly important if the sample has not previously been subjected to any lysing conditions and can therefore, for example, still contain intact viruses etc. In order to enable quantitative isolation of the entire viral nucleic acids, the viral particles must first be destroyed by adding the lysing component. The amount of lysing component required is known to the person skilled in the art.

In a particularly preferred embodiment, the binding buffer additionally contains one or more sulfate salts, such as cobalt sulfate, zinc sulfate or preferably ammonium sulfate or potassium sulfate. It has been found that this additive improves the binding and thus also the isolation of RNA in particular. The concentration of the sulfate salts in the mixture of binding buffer and sample is preferably below 300 mM. The binding buffer particularly preferably contains between 100 and 200 mM ammonium sulfate.

The carrier material with the nucleic acids bound to it is then washed at least once in a next step with at least one washing buffer. The pH value of the wash buffer is typically between 4 and 7, preferably between 4.5 and 6.5. The buffer concentration can be lower than with the binding buffer; it should be in the range of 1 to 50 mM, preferably about 5 to 15 mM. If appropriate, one of the washing buffers can contain additives, for example chelating agents such as EDTA, chaotropic substances and / or nonionic detergents. The washing buffers disclosed according to the invention do not elute the bound nucleic acids, even if they have been adsorbed onto the carrier material with the addition of chaotropic substances. In a washing buffer according to the present invention, additions of organic solvents and / or chaotropic substances are not necessary in order to avoid the premature elution of nucleic acids. According to the invention, an essential component of the washing buffer is at least one component that degrades proteins, such as a proteinase. It has been found that the effectiveness of nucleic acid isolation can be greatly improved by adding such a component to the washing buffer. Proteinase K is particularly preferably added to the washing buffer. Typically, the wash buffer contains 0.1 to

4 mg / ml of an enzyme such as Proteinase K, particularly preferably between 0.2 and 1 mg / ml.

The elution, i.e. the detachment of the bound nucleic acids from the carrier material then takes place by simply increasing the pH to above 7.5. The elution buffer used for this purpose should maintain a pH range of 7.5 to 9, preferably 8 to 8.5. Suitable buffers are e.g. Tris-HCl buffer, Tricin, Bicin and other buffers that buffer in this pH range, preferably Tris / HCl. The buffer concentration should

5 to 10 mM, preferably about 8 to 10 mM. The elution buffer can optionally contain chelating agents such as EDTA and / or other inhibitors of nucleases.

In a preferred embodiment, the volume of the elution buffer is less than 1/10 of the volume of the original used liquid sample. In this way, the isolated nucleic acids are simultaneously concentrated.

The eluted nucleic acids are directly, without further purification steps, for molecular biological applications, e.g. for amplification reactions, can be used.

The method according to the invention for the isolation and purification of nucleic acids is typically carried out in such a way that e.g. a plasma containing the nucleic acids is mixed with the binding buffer and placed in a sample tube. The sample tube may already contain the preferred carrier material. The carrier material can also be added after the liquid sample has been filled in. After an incubation period of typically 1 to 10 minutes, the carrier-nucleic acid complex is separated from the supernatant and the supernatant is discarded. It is resuspended with a washing buffer, separated again and the supernatant is discarded again. Several of these washing steps can optionally be carried out in succession with washing buffers of different compositions. Accordingly, a reagent assembly according to the invention, i.e. a test kit, also containing several wash buffers. Finally, the elution buffer is added to the support material-nucleic acid complex and, after the support material has been separated off, the supernatant containing the nucleic acids is transferred to a new empty tube. This eluate can then be used directly for further analysis methods (PCR, NASBA). When using e.g. Corresponding magnetic particles, the centrifugation frequently used to remove the supernatant can be replaced by the application of a magnetic field.

For an effective washing of the carrier material-nucleic acid complex and for an effective elution of the nucleic acids from the carrier material, it is important when using particulate carrier materials that the Carrier material is homogeneously resuspended in the buffers after separation. In a preferred embodiment, when the washing buffer and / or the elution buffer are added, one or more balls are therefore additionally added. These balls consist of solid materials which are inert in the buffers, preferably of glass, a hard plastic or metal. The material of the spheres does not interact with nucleic acids. The balls are larger than the particles of the carrier material, preferably between 100 and 1000 times larger. When the reaction vessel is shaken gently, these balls result in a homogeneous distribution of the carrier material in the buffer solution and this process step is very easy to automate. In particular, the carrier material is effectively removed from the vessel wall and clumps are dissolved. The addition of the balls has proven to be particularly effective when using magnetizable carrier materials and in automation processes.

The present invention furthermore relates to a test kit for carrying out the method according to the invention. The test kit contains at least one solid phase, preferably a silica solid phase, a binding buffer and a washing buffer, which contains at least one component that degrades proteins. Other optional components include:

- one or more additional wash buffers

an elution buffer to detach the bound nucleic acids from the support material

one or preferably several balls in a suitable dosage form, e.g. in buffer or water, which are used for effective resuspension of the carrier material in washing and / or elution buffer

- Instructions for performing the method according to the invention The preferred compositions of the reagents mentioned in the description of the method according to the invention also apply to the components of the test kit according to the invention.

The present invention makes it possible to extract and enrich a small number of viral genome copies from a large volume, such as a pool of donor plasmas, with relatively simple and inexpensive manipulations, so that the subsequent detection reaction provides reliable results. With magnetic silica particles, the entire process can automatically extract highly pure viral nucleic acids from many primary tubes in less than an hour in a high-throughput process and replaces the preconcentration process.

For the first time, the method according to the invention opens up the possibility of using a very small number of copies (<50 copies) of e.g. Detect viruses in liquid samples. In blood banks, several individual samples of body fluids, such as plasma or serum, are often combined to form so-called sample pools. In order that small copy numbers of viruses can also be detected in this sample pool, it is usually not sufficient to examine a sample of 100 μl. With the method according to the invention, copy numbers of e.g. among 50 viruses can be detected in large-volume samples over 2 ml. In addition, the elution volume after isolation of the nucleic acids is preferably less than 1/10 of the sample volume originally used, so that at the same time a large reduction in the sample volume and thus a concentration of the nucleic acids is possible.

Even without further explanations, it is assumed that a person skilled in the art can use the above description in the broadest scope. The preferred embodiments and examples are therefore only as descriptive disclosure, which is in no way to be construed as limiting in any way.

The complete disclosure of all applications, patents and publications mentioned above and below, and the corresponding application DE 102 58258.0, filed on December 13, 2002, are incorporated by reference into this application.

Examples

Example 1, basic protocol S for small plasma samples

1.5 mg MagPrep ^® silica particles (Merck KGaA) are resuspended in 900 // I binding buffer TAAN (200mM acetate-Tris pH 4.0, 0.5% NP40, 200mM ammonium sulfate). This binding buffer mix also contains internal control RNA and / or DNA.

100μl plasma sample are mixed homogeneously with 900 / I binding buffer mix. After incubation for 1-10 minutes, magnetization is carried out and the supernatant is carefully separated. After removal of the magnetic field, the particles are resuspended with 500 I wash buffer (10mM Tris-HCl pH 6.6; 0.5mg / ml Proteinase K) and incubated for up to 10 minutes at room temperature.

After magnetization, the supernatant is carefully removed and 500 / l washing buffer is added again. After removing the magnetic field, the particles are resuspended, magnetized and the supernatant discarded. The nucleic acids are eluted from the particles after incubation for 10 minutes in 100 tl of elution buffer (10 mM Tris / HCl pH 8.5) at 80 ° C. After magnetization, the eluate is transferred to a new, sterile vessel. Example 2, basic protocol XL for pools and large-volume incremental samples

A plasma pool is produced in which 0.1 ml of 24 plasma samples are taken and combined in a suitable sample tube. One of the plasma samples contains virus particles. 7.5 mg MagPrep ^® silica particles are resuspended in 7.6 ml binding buffer TA (200mM acetate-Tris pH 4.0, 0.5% NP40, 200mM ammonium sulfate). The binding buffer TA also contains internal control RNA and / or DNA. The 2.4 ml of the plasma pool are mixed homogeneously with a 7.6 ml binding buffer mix. After incubation for 1-10 minutes, magnetization is carried out and the supernatant is carefully separated. After removal of the magnetic field, the particles are resuspended with 5 ml washing buffer (1 OmM Tris-HCl pH 6.6; 0.5 mg / ml Proteinase K) and incubated for up to 10 minutes at room temperature.

After magnetization, the supernatant is carefully removed and 5 ml of washing buffer are added again. After removing the magnetic field, the particles are resuspended, magnetized and the supernatant discarded. The nucleic acids are eluted from the particles after 10 minutes of incubation in 10O / - / I elution buffer (10mM Tris / HCl pH 8.5) at 80 ° C. After magnetization, the eluate is transferred to a new, sterile vessel.

The nucleic acids of the eluates obtained are amplified by (RT) PCR and appropriate gene probes and detected (eg, using a thermal cycler such as the ABI Prism 7000 (TaqMan ^®) manufactured by Applied Biosystems or the LightCycler ^® from Roche).

Example 3) Influence of large sample volumes on the sensitivity

The results of this example are shown graphically in Figure 1. The Y axis shows the yield of the copy number of the viruses in%. The 4 approaches explained below are listed on the X axis. 100 / I virus-positive plasma (mixed with 600 copies of HIV: striped bars and 200 copies of HBV: dotted bars - in Figure 1) were either extracted directly (sample A and Q) or were treated with 2.3 ml (sample 24P) or 4.7 ml (sample 48P) virus-free plasma mixed and the entire large volume extracted. For sample A the basic protocol S according to example 1 was used, for the samples 24P and 48P the basic protocol XL according to example 2 was used. Sample Q was extracted in accordance with the QIA & Viral RNA Extraction Kit from QIAGEN. Found for sample A sensitivities (copy number in the eluates detected in quantitative TaqMan ^® PCR) equal to 100% has been set.

As Figure 1 shows, despite a 23-fold or 47-fold dilution of the virus-positive sample with virus-free plasma, the few viral DNAVRNA molecules from the pool can be detected quantitatively with almost no loss.

Example 4) Effect of sulfate salts on the isolation of RNA

100 / I of HBV- and HIV-positive plasma (approx. 1000 copies each) are incubated with MagPrep ^® silica particles and 900 / I of a binding buffer, which either contains no ammonium sulfate (sample # 1) or 200mM ammonium sulfate (sample # 2) , The further isolation takes place according to example 1.

The results of this experiment are shown in Figure 2. The yield of nucleic acids is given in% on the Y axis. The binding of 100% corresponds to the signal strength of the quantitative amplification of 1000 molecules of purified HBV-DNA or HIV-RNA. On the X axis approaches 1 and 2 explained above are plotted. (HBV: hatched bar; HIV: gray bar)

It clearly shows that the yield of RNA molecules can be improved to the desired sensitivity by adding ammonium sulfate to the binding buffer.

Example 5) Effect of adding Proteinase K

Four 100 μl plasma samples are provided. A plasma sample is pre-incubated with Proteinase K at a concentration of 0.5mg / ml for 4 hours (Sample # 1). Then virus particles (HIV and HBV; 1000 copies / ml each) are added to all four plasma samples. The four samples are extracted analogously to basic protocol S (example 1), with the washing buffer containing no proteinase K in sample # 1, # 2 and # 3. In sample # 3, 0.5 mg / ml Proteinase K is added to the binding buffer. In sample # 4, 0.5 mg / ml Proteinase K is added to the wash buffer.

The results of this experiment are shown in Figure 3. The yield of nucleic acids is given in% on the Y axis. The sensitivity found for sample # 3 (number of copies in the eluates detected in the quantitative TaqMan ^® PCR) is set to 100%. The four approaches 1 to 4 explained above are plotted on the X axis.

The results show that the desired effectiveness of the isolation of the nucleic acids can only be achieved when the proteinase is added to the washing buffer.

The addition of Proteinase K to the binding buffer or the pre-incubation of the sample with Proteinase K shows no significant improvement. Example 6) optimal concentration of the carrier material for large and small sample volumes

Each 100 / I HIV-positive plasma (approx. 1000 copies) is either extracted according to basic protocol S (example 1) (striped bars) or mixed with 2.3 ml virus-free plasma and extracted according to basic protocol XL (example 2) ( gray bars). MagPrep ^® silica particles are used as the carrier material. The amount of the carrier material is varied. The amount used per ml of plasma is shown in Figure 4 on the X axis.

The yield of isolated nucleic acids is given in% on the Y axis. The yield for 15 mg / ml was set to 100%.

The results show that when using large sample volumes over 2 ml, the amount of carrier material can be reduced in relation to the amount required for small sample volumes. Furthermore, it becomes clear that the amount of the carrier material used has less influence on the yield in the case of large sample volumes. A lower concentration of carrier material of about 2 to 3 mg / ml leads to slightly better yields than twice the amount of approx. 6 mg / ml.

Claims

Expectations

1. A method for isolating nucleic acids from liquid samples characterized by the following method steps: a) providing a liquid sample which contains nucleic acids; b) providing a carrier material made of an inorganic hydroxyl-containing oxidic material; c) mixing the sample from step a) with a binding buffer so that the pH of the mixture is below 7; d) treating the sample from step c) acidified by means of a binding buffer with the carrier material from step b), the nucleic acids being bound to the carrier material; e) separating the carrier material with the bound nucleic acids from the rest of the sample and the binding buffer; f) washing the carrier material with a washing buffer which contains at least one component which degrades proteins; g) Elution of the nucleic acids bound in step d) from the support material with an alkaline elution buffer.

2. The method according to claim 1, characterized in that the liquid sample provided in step a) consists of cell-free body fluid.

3. The method according to any one of claims 1 or 2, characterized in that the binding buffer additionally contains at least one lysing component.

4. The method according to one or more of claims 1 to 3, characterized in that the binding buffer additionally contains at least one sulfate salt.

5. The method according to one or more of claims 1 to 4, characterized in that the liquid sample provided in step a) has a volume of over 2 ml.

6. The method according to one or more of claims 1 to 5, characterized in that the liquid sample provided in step a) consists of a mixture of at least 10 individual samples.

7. The method according to one or more of claims 1 to 6, characterized in that the volume of the elution buffer in step g) is less than 1/10 of the volume of the sample provided in step a).

8. The method according to one or more of claims 1 to 7, characterized in that in step f) and / or step g) one or more balls are added to support the resuspension of the carrier material.

9. Test kit for carrying out the method according to one or more of claims 1 to 8 at least comprising a carrier material made of an inorganic hydroxyl-containing oxidic material, a binding buffer and a washing buffer which contains at least one component which degrades proteins.

10. Use of the method according to one or more of claims 1 to 8 for the isolation of viral nucleic acids from body fluids.