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EP1570077A1 - Procede de purification d'acides nucleiques - Google Patents

Procede de purification d'acides nucleiques

Info

Publication number
EP1570077A1
EP1570077A1 EP03779999A EP03779999A EP1570077A1 EP 1570077 A1 EP1570077 A1 EP 1570077A1 EP 03779999 A EP03779999 A EP 03779999A EP 03779999 A EP03779999 A EP 03779999A EP 1570077 A1 EP1570077 A1 EP 1570077A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acids
buffer
sample
carrier material
binding buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03779999A
Other languages
German (de)
English (en)
Inventor
Uwe Michelsen
Willi Roth
Kai Hourfar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Patent GmbH
Original Assignee
Merck Patent GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Patent GmbH filed Critical Merck Patent GmbH
Publication of EP1570077A1 publication Critical patent/EP1570077A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • 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.
  • nucleic acids from complex biological starting materials, such as body fluids, is of great importance e.g. for the detection of viral diseases.
  • nucleic acid amplification techniques e.g. Polymerase chain reaction (PCR), branched DNA detection (bDNA) or nucleic acid sequence based amplification (NASBA) are used.
  • PCR Polymerase chain reaction
  • bDNA branched DNA detection
  • NASBA nucleic acid sequence based amplification
  • HBV Hepatitis B or C virus
  • HCV human immunodeficiency virus
  • DNA or RNA viral nucleic acids
  • 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).
  • 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.
  • the DNA or RNA is extracted and suitably purified for the detection reaction.
  • 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.
  • 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.
  • the liquid sample provided in step a) consists of cell-free body fluid.
  • the binding buffer additionally contains at least one lysing component, such as a non-ionic detergent.
  • the binding buffer additionally contains at least one sulfate salt, e.g. Ammonium sulfate, potassium sulfate, cobalt sulfate or zinc sulfate.
  • sulfate salt e.g. Ammonium sulfate, potassium sulfate, cobalt sulfate or zinc sulfate.
  • the liquid sample provided in step a) has a volume of over 2 ml.
  • 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 ,
  • the volume of the elution buffer in step g) is less than 1/10 of the volume of the sample provided in step a).
  • 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 ⁇ l 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.
  • 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).
  • silica particles MagPrep ® Merck KGaA, Darmstadt.
  • One of the binding buffers mentioned below is preferably used as the equilibration buffer for the carrier material.
  • components that break down proteins are, in particular, enzymes such as proteinases, particularly preferably proteinase K.
  • 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.
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • 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.
  • a binding buffer 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.
  • a binding buffer with a concentration of 100-200 mM 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.
  • the binding buffer particularly preferably contains neither ionic detergents nor chaotropic substances in concentrations> 500 mM, preferably> 200 mM.
  • 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.
  • the viral particles 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the wash buffer contains 0.1 to
  • 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 be used for this purpose.
  • the elution buffer can optionally contain chelating agents such as EDTA and / or other inhibitors of nucleases.
  • 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.
  • 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.
  • 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).
  • PCR e.g. Corresponding magnetic particles
  • the centrifugation frequently used to remove the supernatant can be replaced by the application of a magnetic field.
  • the Carrier material is homogeneously resuspended in the buffers after separation.
  • 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.
  • 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:
  • 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
  • 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.
  • a large volume such as a pool of donor plasmas
  • 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.
  • 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.
  • 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 ⁇ l.
  • copy numbers of e.g. among 50 viruses can be detected in large-volume samples over 2 ml.
  • 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.
  • 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.
  • 100 ⁇ l 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.
  • 500 I wash buffer (10mM Tris-HCl pH 6.6; 0.5mg / ml Proteinase K)
  • 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.
  • 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).
  • 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.
  • sample A the basic protocol S according to example 1 was used
  • 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.
  • Example # 1 Four 100 ⁇ l 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.
  • 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%.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant d'isoler des acides nucléiques à partir d'échantillons biologiques, notamment d'échantillons de grand volume, à l'aide d'un matériau support inorganique. La liaison des acides nucléiques au matériau support s'effectue dans des conditions acides, l'élution s'effectuant dans des conditions basiques. Les rendements obtenus des acides nucléiques sont considérablement augmentés, du fait qu'une protéinase est ajoutée au tampon de lavage.
EP03779999A 2002-12-13 2003-11-20 Procede de purification d'acides nucleiques Ceased EP1570077A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE2002158258 DE10258258A1 (de) 2002-12-13 2002-12-13 Verfahren zur Aufreinigung von Nukleinsäuren
DE10258258 2002-12-13
PCT/EP2003/012977 WO2004055207A1 (fr) 2002-12-13 2003-11-20 Procede de purification d'acides nucleiques

Publications (1)

Publication Number Publication Date
EP1570077A1 true EP1570077A1 (fr) 2005-09-07

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ID=32336278

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Application Number Title Priority Date Filing Date
EP03779999A Ceased EP1570077A1 (fr) 2002-12-13 2003-11-20 Procede de purification d'acides nucleiques

Country Status (4)

Country Link
EP (1) EP1570077A1 (fr)
AU (1) AU2003288124A1 (fr)
DE (1) DE10258258A1 (fr)
WO (1) WO2004055207A1 (fr)

Families Citing this family (12)

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Publication number Priority date Publication date Assignee Title
JP4811773B2 (ja) * 2005-06-08 2011-11-09 肇 柄谷 核酸の精製法
DE102005057334A1 (de) 2005-11-28 2007-06-06 Aj Innuscreen Gmbh Verfahren zur Isolierung von Nukleinsäuren aus beliebigen Ausgangsmaterialien
DE102005059217B4 (de) * 2005-12-07 2011-03-17 Aj Innuscreen Gmbh Verfahren und Testkit zur Trennung, Aufreinigung und Wiedergewinnung von lang- und kurzkettigen Nukleinsäuren
KR100785010B1 (ko) * 2006-04-06 2007-12-11 삼성전자주식회사 수소 결합을 이용하여 고체 지지체의 친수성 표면 상에서핵산 정제 방법 및 장치
DE102006032610C5 (de) * 2006-07-11 2016-09-15 Aj Innuscreen Gmbh Verfahren zur parallelen Isolierung viraler Nukleinsäuren
US10131935B2 (en) 2006-07-11 2018-11-20 Aj Innuscreen Gmbh Method for parallel isolation of viral nucleic acids
DE102007035250A1 (de) * 2007-07-27 2009-01-29 Qiagen Gmbh Verfahren zum Abtrennen von nicht-proteinhaltigen Biomolekülen, insbesondere Nukleinsäuren aus proteinhaltigen Proben
GB0814570D0 (en) * 2008-08-08 2008-09-17 Diagnostics For The Real World Isolation of nucleic acid
CA2773186A1 (fr) 2009-09-24 2011-03-31 Qiagen Gaithersburg, Inc. Compositions, procedes, et kit d'isolement et d'analyse d'acides nucleiques en utilisant un materiau echangeur d'anions
DE102014111210B3 (de) 2014-08-06 2016-01-07 GFE Blut mbH Verfahren und Vorrichtung zum automatisierten Bearbeiten von gepoolten Proben
WO2016073824A1 (fr) * 2014-11-07 2016-05-12 The Johns Hopkins University Méthode sans agents chaotropiques ni composés volatils pour la purification d'acides nucléiques issus de plasma
EP3502274A1 (fr) * 2017-12-22 2019-06-26 Attomol GmbH Support d'échantillon et procédé d'obtention dudit support

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EP0818461B1 (fr) * 1996-07-12 2005-09-28 Toyo Boseki Kabushiki Kaisha Procédé pour isoler des acides ribonucléiques.
EP0897978A3 (fr) * 1997-08-22 2001-10-17 Becton, Dickinson and Company Oxyde de zirconium et de composés apparentés pour la purification des acides nucléiques
EP1071691B1 (fr) * 1998-02-04 2005-09-28 MERCK PATENT GmbH Procede pour isoler et purifier des acides nucleiques
DE19836559A1 (de) * 1998-08-12 2000-03-23 Antigen Gmbh Gefäß zur Entnahme von Blut
DE10157624A1 (de) * 2001-11-26 2003-06-05 Merck Patent Gmbh Verfahren zur Anreicherung und zum Nachweis von HIV

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Also Published As

Publication number Publication date
WO2004055207A1 (fr) 2004-07-01
AU2003288124A1 (en) 2004-07-09
DE10258258A1 (de) 2004-06-24

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