WO2008006865A1 - Procédé d'isolation d'acides nucléiques viraux - Google Patents

Procédé d'isolation d'acides nucléiques viraux Download PDF

Info

Publication number
WO2008006865A1
WO2008006865A1 PCT/EP2007/057131 EP2007057131W WO2008006865A1 WO 2008006865 A1 WO2008006865 A1 WO 2008006865A1 EP 2007057131 W EP2007057131 W EP 2007057131W WO 2008006865 A1 WO2008006865 A1 WO 2008006865A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acids
double
binding
nucleic acid
detergent
Prior art date
Application number
PCT/EP2007/057131
Other languages
German (de)
English (en)
Inventor
Timo Hillebrand
Original Assignee
Aj Innuscreen 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
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=38544123&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2008006865(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Aj Innuscreen Gmbh filed Critical Aj Innuscreen Gmbh
Priority to EP07787402A priority Critical patent/EP2041310A1/fr
Publication of WO2008006865A1 publication Critical patent/WO2008006865A1/fr

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 method for fast, simple and highly sensitive isolation of viral nucleic acids.
  • US Pat. No. 5,234,809 describes a process for isolating nucleic acids from nucleic acid-containing starting materials by incubating the starting material with a chaotropic buffer and a DNA-binding solid phase.
  • the then binding solid phase consists of silicate mineral particles, which are> 50 nm.
  • the chaotropic buffers realize both the lysis of the starting material and the binding of the nucleic acids to the solid phase.
  • the method is well suited for isolating nucleic acids from small sample quantities and finds its practical application especially in the field of isolation of viral nucleic acids.
  • the method described in the patent has a number of disadvantages.
  • the method realizes efficient isolation and possibly also separation of nucleic acids / nucleic acid mixtures and is easy and quick to carry out.
  • the improvement over the patent cited above is achieved by the addition of an alcohol to the initial lysis buffer. This step makes it possible to increase the efficiency of the extraction process, which also allows sensitive nucleic acid targets to be isolated from a clinical sample.
  • Another method of separating and isolating single-stranded and double-stranded nucleic acids is disclosed in the patent EP 1 146049 A2.
  • the method is based on the treatment of a source containing nucleic acids with at least one mineral carrier material in the form:
  • the single-stranded nucleic acid is isolated by adjusting the treatment conditions by an aqueous mixture of chaotropic salts and lower aliphatic alcohols, such that subsequently predominantly the single-stranded nucleic acid is adsorbed to a mineral carrier, the double-stranded nucleic acid, however, not adsorbed.
  • the bound single-stranded nucleic acid is subsequently washed and finally eluted from the support by means of a low salt buffer.
  • the double-stranded nucleic acid is isolated by adjusting the treatment conditions by a mixture of alkaline earth ion-complexing substances without lower aliphatic alcohols, such that subsequently predominantly the double-stranded nucleic acid is adsorbed to a mineral carrier, the single-stranded
  • nucleic acid however, not adsorbed.
  • the bound double-stranded nucleic acid is subsequently washed and finally eluted from the support by means of a low salt buffer.
  • the double-stranded nucleic acid is isolated by adjusting the treatment conditions by the presence of a sarcosinate but without lower aliphatic alcohols, such that subsequently predominantly the double-stranded nucleic acid is adsorbed to a mineral carrier, the single-stranded nucleic acid, however, does not adsorb.
  • the bound double-stranded nucleic acid is subsequently washed and finally eluted from the support by means of a low salt buffer.
  • the double-stranded or single-stranded nucleic acids is isolated by adjusting the treatment conditions by an aqueous mixture of chaotropic salts and lower aliphatic alcohols, such that both nucleic acid fractions adsorb to a mineral carrier.
  • the separation of the nucleic acids takes place by a selective elution.
  • the double-stranded nucleic acid is detached from the carrier material by means of a solution of reduced ionic strength and lower aliphatic alcohols.
  • the remaining single-stranded nucleic acid is subsequently washed and finally detached from the carrier material by means of a low-salt buffer.
  • the single-stranded nucleic acid can be selectively eluted from the support material by means of a solution containing alkaline earth metal complexing substances and / or sarcosinates.
  • the now remaining double-stranded nucleic acid is subsequently washed again and finally detached from the carrier material by means of a low-salt buffer.
  • a lysis buffer which contains 5% Triton and no reference to an alcohol.
  • a buffer is used, which is composed of 6M guanidine hydrochloride, 1OmM Tris HCl and 20% Triton X-100. This buffer is added to the biological sample. This is followed by the addition of proteinase K. It is then followed by incubation at 70 0 C.
  • this buffer in combination with the proteinase K fulfills the function of a lysis buffer.
  • a further component is added to the batch (eg isopropanol, acetonitrile, DMSO or methyl ethyl ketone), which is a water-miscible, non-organic, organic component.
  • N-methyl-2-pyrrolidone or, alternatively, isopropanol is used as the "non-acidic" organic component.
  • this also concerns the isolation of DNA by means of the binding of the nucleic acid to magnetic particles.
  • the addition of N-methyl-2-pyrrolidone shows similar yields as the addition of isopropanol.
  • Example 4 uses a lysis buffer, which in turn after lysis of the sample, the addition of a binding conditioner (gamma-butyl lactone, propylene carbonate or turn N-methyl-2-pyrrolidone) takes place.
  • a binding conditioner gamma-butyl lactone, propylene carbonate or turn N-methyl-2-pyrrolidone
  • the detergent component which is listed in EP 1 524317 Al (Triton X-100), is only part of the lysis buffer. However, there is no indication that this component mediates binding of DNA or RNA. According to the patent specification, this is realized by the additional addition of the non-acidic organic components according to the invention. It turns out that the lysis buffer is not able to realize isolation of nucleic acids. For the isolation of DNA or RNA, the listed organic component must be added.
  • the components listed in EP 1 524317 Al are also highly toxic (eg DMSO) and represent a significant hazard.
  • the invention was based on the object to eliminate the disadvantages mentioned in the prior art.
  • the object of the invention could be solved in a surprising manner.
  • the method is characterized by claim 1 with its alternative methods.
  • the dependent claims 2 to 8 relate to specific embodiments of the method according to the invention.
  • an alternative method has been provided, which allows to quickly, easily and inexpensively isolate nucleic acids from a sample containing nucleic acid, with the necessary solutions for the selective binding of nucleic acids - with the exception of detergents - no further water-miscible non-acidic organic components , such as alcohol, acetonitrile, DMSO or methyl ethyl ketone.
  • the method makes it possible to isolate DNA (double-stranded nucleic acid) and RNA (single-stranded nucleic acid) from a starting sample at the same time and thus does not cause separation of nucleic acid mixtures.
  • the method is able to isolate highly sensitive viral nucleic acids from diagnostically relevant biological samples.
  • EDTA and sodium citrate as salts of organic acids do not belong to the water-miscible non-acidic organic components.
  • the inventive method is based on the lysis of a biological sample with known lysis buffers.
  • the lysis buffers contain, for example, known chaotropic salts of high ionic strengths or combinations of chaotropic salts of high ionic strengths with a sarcosinate and optionally further additives. These buffer systems allow in their known function the digestion and denaturation of a biological sample and also cause inactivation of endogenous RNases. This is especially important with regard to the isolation of RNA.
  • lysis buffers which are a combination of chaotropic salts and other salts and which, if appropriate, also contain detergents and optionally further additives in support of the lysis process, with the chaotropic salts not requiring any high ionic strengths known per se.
  • proteolytic enzymes eg proteinase K
  • the binding conditions for binding the nucleic acids contained in the sample are then adjusted to a mineral carrier material.
  • a nonionic detergent such as. Tween-20, Tween-80 or Triton X-100 in a high concentration.
  • the addition of the detergent is the essential step. Setting an acidic pH is advantageous.
  • the detergent completely replaces the function of adding a previously necessary alcoholic component.
  • the lysis mixture which is mixed with the detergent, is subsequently brought into contact with a carrier, preferably a mineral carrier or surface-functionalized magnetic particles or iron oxide particles, whereby the nucleic acids to be isolated are adsorbed to the mineral carrier.
  • the mineral carrier is subsequently washed with known washing buffers and final, in turn, detached from the mineral material by means of water or a low salt buffer.
  • the detergent component required for the binding of the nucleic acids can also be present as a mixture of detergent with further salts and optionally further additives.
  • optimal binding conditions in the combination of salt / detergent can always be achieved without this having an effect on the initial use of an efficient lyophil buffer.
  • a lysis buffer for initial sample digestion are used, which has only a low salt concentration (which would not be sufficient to provide in combination with a detergent efficient binding of the nucleic acids to be isolated).
  • a binding buffer is then added to the reaction mixture which has a nonionic detergent of high concentration and, in addition, a high salt concentration. This then sets the binding conditions that efficiently mediate quantitative isolation of the viral nucleic acids. It is always crucial that the binding buffer based on a high concentration of nonionic detergents also provides the further components which enable an efficient binding of the nucleic acids to be isolated.
  • the method is thus completely alternative to the described methods which require an alcohol or other substances such as acetonitrile, DMSO or methyl ethyl ketone for efficient attachment of nucleic acids to mineral support materials. It moreover permits the parallel isolation of double-stranded and single-stranded nucleic acids from a biological sample without separating the double-stranded and single-stranded nucleic acids.
  • a decisive advantage is that a high degree of flexibility is achieved with regard to the selection of the lysis buffer.
  • well-known, efficiently acting lysis buffer including proteolytic enzymes can be used.
  • the adjustment of the binding conditions for nucleic acids for their adsorption to a mineral carrier material is always carried out after the lysis of the starting material by the addition of the binding buffer according to the invention, containing a high concentration of a nonionic detergent and optionally containing further additives.
  • the invention allows for the first time the parallel isolation of double-stranded and single-stranded nucleic acids from a nucleic acid-containing sample, wherein the nucleic acid-containing sample with a buffer consisting of either a salt solution of chaotropic salts or a salt solution of chaotropic salts of low ionic strength (less than 100 mM) and others nonchaotropic salts, and optionally other additives such as detergents (SDS, sarcosinate, LDS), proteolytic enzymes, complexing compounds (EDTA, EGTA) is incubated under these conditions, a digestion of the biological sample is carried out so that the nucleic acids in the buffer be released
  • binding buffer consisting of:
  • a mineral material carrier
  • concentration of detergent for binding the nucleic acids is 5 - 50%; preferably 10 -30% in the final mixture of lysis buffer / binding buffer.
  • the mineral material is subsequently washed with washing buffer and the nucleic acids by means of water or low salt buffer from the mineral material again dissolves.
  • kits for carrying out the method according to the invention which is described in claims 9 to 11.
  • the inventive use is that nonionic detergents in a concentration of 5 - 50%; preferably 10 -30%, in the absence of alcohol, for parallel isolation of single and double-stranded nucleic acids from samples containing these substances without separating the double- and single-stranded nucleic acids by binding the total nucleic acids to a solid support.
  • the invention is particularly advantageous in the isolation of viral nucleic acids.
  • Two blood plasma samples were spiked with HIV viruses and HBV viruses and used for the isolation of the viral nucleic acid.
  • 150 ⁇ l of the sample was transferred to a 1.5 ml reaction vessel.
  • the sample was subsequently added with 450 ⁇ l of a lysis buffer (4 M guanidine thiocyanate, 80 mM trisodium citrate dihydrate) and vortexed for 10 s. Subsequently, the sample was incubated at room temperature for 10 min. After lysis and denaturation of the starting sample, 600 ⁇ l of a binding buffer (30% Tween-20, 5 mM EDTA, 20 mM trisodium citrate dihydrate) was added and the sample completely mixed.
  • a binding buffer (30% Tween-20, 5 mM EDTA, 20 mM trisodium citrate dihydrate
  • the binding buffer is adjusted to an acidic pH, for example by means of an acetate buffer.
  • the batch was subsequently centrifuged over a filter column which contained a commercially available glass fiber filter paper (Whatman).
  • the filter column was subsequently washed with alcoholic wash buffers. Centrifuged briefly to dry the filter material.
  • the elution of the bound viral nucleic acid was carried out by the addition of 50 ⁇ l RNAse free water.
  • the isolated nucleic acid was then analyzed for specific virus detection of HIV or HBV by means of real-time PCR. The detection of viral nucleic acids was successful.

Landscapes

  • 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 d'isolation parallèle d'acides nucléiques double brin et simple brin à partir d'échantillons contenant ces matières, sans séparation des acides nucléiques double brin et simple brin, procédé selon lequel les échantillons sont déplacés au moyen de tampons de lyse connus (hautes concentrations de sel ou faibles concentrations de sel ou avec des enzymes protéolytiques). L'échantillon contenant les acides nucléiques est ajusté avant lyse, après lyse ou après homogénéisation, au moyen d'un tampon de liaison acide contenant au moins un détergent non ionique en haute concentration, de sorte que l'ensemble de l'acide nucléique est adsorbé sur un support solide.
PCT/EP2007/057131 2006-07-11 2007-07-11 Procédé d'isolation d'acides nucléiques viraux WO2008006865A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP07787402A EP2041310A1 (fr) 2006-07-11 2007-07-11 Procédé d'isolation d'acides nucléiques viraux

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006032610.5A DE102006032610C5 (de) 2006-07-11 2006-07-11 Verfahren zur parallelen Isolierung viraler Nukleinsäuren
DE102006032610.5 2006-07-11

Publications (1)

Publication Number Publication Date
WO2008006865A1 true WO2008006865A1 (fr) 2008-01-17

Family

ID=38544123

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/057131 WO2008006865A1 (fr) 2006-07-11 2007-07-11 Procédé d'isolation d'acides nucléiques viraux

Country Status (3)

Country Link
EP (1) EP2041310A1 (fr)
DE (1) DE102006032610C5 (fr)
WO (1) WO2008006865A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018731A2 (fr) * 1994-12-12 1996-06-20 Dynal A/S Isolement de l'acide nucleique
WO1999040098A1 (fr) * 1998-02-04 1999-08-12 Merck Patent Gmbh Procede pour isoler et purifier des acides nucleiques
DE19856064A1 (de) * 1998-12-04 2000-06-29 Invitek Gmbh Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien
WO2003046213A2 (fr) * 2001-11-26 2003-06-05 Merck Patent Gmbh Procede pour concentrer et identifier des vih
DE10253351A1 (de) * 2002-11-08 2004-05-27 InViTek Gesellschaft für Biotechnik & Biodesign mbH Neuartige Pufferfomulierungen zur Isolierung, Reinigung und Rückgewinnung lang- und kurzkettiger Nukleinsäuren
WO2004055207A1 (fr) * 2002-12-13 2004-07-01 Merck Patent Gmbh Procede de purification d'acides nucleiques
EP1524317A1 (fr) * 2003-10-13 2005-04-20 Boehringer Mannheim Gmbh Procédé pour l'isolation d'acides nucléiques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA05001815A (es) * 2004-02-20 2005-08-24 Hoffmann La Roche Adsorcion de acidos nucleicos a una fase solida.

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018731A2 (fr) * 1994-12-12 1996-06-20 Dynal A/S Isolement de l'acide nucleique
WO1999040098A1 (fr) * 1998-02-04 1999-08-12 Merck Patent Gmbh Procede pour isoler et purifier des acides nucleiques
DE19856064A1 (de) * 1998-12-04 2000-06-29 Invitek Gmbh Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien
WO2003046213A2 (fr) * 2001-11-26 2003-06-05 Merck Patent Gmbh Procede pour concentrer et identifier des vih
DE10253351A1 (de) * 2002-11-08 2004-05-27 InViTek Gesellschaft für Biotechnik & Biodesign mbH Neuartige Pufferfomulierungen zur Isolierung, Reinigung und Rückgewinnung lang- und kurzkettiger Nukleinsäuren
WO2004055207A1 (fr) * 2002-12-13 2004-07-01 Merck Patent Gmbh Procede de purification d'acides nucleiques
EP1524317A1 (fr) * 2003-10-13 2005-04-20 Boehringer Mannheim Gmbh Procédé pour l'isolation d'acides nucléiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOOM R. ET AL.: "RAPID AND SIMPLE METHOD FOR PURIFICATION OF NUCLEIC ACIDS", JOURNAL OF CLINICAL MICROBIOLOGY, WASHINGTON, DC, US, vol. 28, no. 3, 1 March 1990 (1990-03-01), pages 495 - 503, XP000607196, ISSN: 0095-1137 *
HOURFAR M.K. ET AL.: "High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation", CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY, WASHINGTON, DC, US, vol. 51, no. 7, July 2005 (2005-07-01), pages 1217 - 1222, XP002403248, ISSN: 0009-9147 *
See also references of EP2041310A1 *

Also Published As

Publication number Publication date
DE102006032610A1 (de) 2008-01-24
EP2041310A1 (fr) 2009-04-01
DE102006032610B4 (de) 2011-05-26
DE102006032610C5 (de) 2016-09-15

Similar Documents

Publication Publication Date Title
EP1851313B1 (fr) Méthode pour l'isolation d'acides nucléiques dans laquelle les acides nucléiques sont immobilisés à haute température sur une matrice
DE69734263T2 (de) Verfahren zur Isolierung von Ribonucleinsäuren.
EP1146049B1 (fr) Procédé de séparation de structures d'acides nucléiques à deux brins et à un brin
EP2285815B1 (fr) Procédé d'isolation d'acides nucléiques à chaînes courtes
WO2011061274A1 (fr) Procédé d'enrichissement et isolement sélectifs d'acides nucléiques microbiens et facultativement en outre viraux
DE102008023297B4 (de) Verfahren zur Anreicherung und Isolierung von Nukleinsäuren oder Viren
EP2446054B1 (fr) Procédé d'amplification d'adn en utilisant du tétraéthylèneglycole
DE112009001513B4 (de) Stabile Lysepuffermixtur zur Extraktion von Nukleinsäuren
EP2041281B2 (fr) Procédé permettant d'isoler en parallèle des acides nucléiques à simple brin et à double brin et de retirer de façon sélective des acides nucléiques à double brin d'un mélange d'acides nucléiques à simple brin et à double brin
DE69632904T2 (de) Kopräzipitat und methode zur extraktion von nukleinsäuren
DE10258258A1 (de) Verfahren zur Aufreinigung von Nukleinsäuren
EP1137800B1 (fr) Procede de purification ou d'isolement d'acides nucleiques viraux
DE10153957A1 (de) Verfahren zur Isolierung von Nukleinsäuren
DE102006032610B4 (de) Verfahren zur parallelen Isolierung viraler Nukleinsäuren
US10131935B2 (en) Method for parallel isolation of viral nucleic acids
WO2003046213A2 (fr) Procede pour concentrer et identifier des vih
EP1709172B1 (fr) Methode et kit d'isolation d'arn
DE102004045332B3 (de) Verfahren zur Isolierung und Trennung von einzel- und doppelsträngigen Nucleinsäuren aus einer diese Nucleinsäuren enthaltenden Probe sowie Trennsäule zur Durchführung dieses Verfahrens
DE102014214173B4 (de) Verfahren zur selektiven größenfraktionierten Isolierung von Nukleinsäuren
EP3215619A1 (fr) Procédé de séparation et d'isolement selon la catégorie dimensionnelle de mélanges d'acides nucléiques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07787402

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: RU

REEP Request for entry into the european phase

Ref document number: 2007787402

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2007787402

Country of ref document: EP