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WO2012007581A1 - Procédé pour enrichir des bactéries, des virus et des cellules, et pour isoler ensuite leurs acides nucléiques - Google Patents

Procédé pour enrichir des bactéries, des virus et des cellules, et pour isoler ensuite leurs acides nucléiques Download PDF

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Publication number
WO2012007581A1
WO2012007581A1 PCT/EP2011/062161 EP2011062161W WO2012007581A1 WO 2012007581 A1 WO2012007581 A1 WO 2012007581A1 EP 2011062161 W EP2011062161 W EP 2011062161W WO 2012007581 A1 WO2012007581 A1 WO 2012007581A1
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WO
WIPO (PCT)
Prior art keywords
cells
viruses
bacteria
nucleic acids
beads
Prior art date
Application number
PCT/EP2011/062161
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
Application filed by Aj Innuscreen Gmbh filed Critical Aj Innuscreen Gmbh
Publication of WO2012007581A1 publication Critical patent/WO2012007581A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • the invention relates to a universal and greatly simplified method for combining accumulation of bacteria, viruses or cells from a sample and the subsequent isolation of the nucleic acid from these targets.
  • the method can be carried out manually or fully automated.
  • the isolation of DNA from cells and tissues is characterized in that the nucleic acid-containing starting materials under strongly denaturing and reducing conditions, partially using protein-degrading enzymes, digested and purified the exiting nucleic acid fractions via phenol / chloroform extraction steps and the nucleic acids are recovered from the aqueous phase by dialysis or ethanol precipitation (Sambrook, J., Fritsch, EF and Maniatis, T., 1989, CSH, "Molecular Cloning").
  • This method has received a number of modifications to date and is currently used for different methods of extracting and purifying nucleic acids from different sources (Marko, MA, Chipperfield, R. and Birnboim, HG, 1982, Anal. Biochem. , 121, 382-387).
  • kits are based on the well-known principle of binding of nucleic acids to mineral carriers in the presence of solutions of different chaotropic salts and use as support materials suspensions finely ground glass powder (eg Glasmilk, BIO 101, La Jolla, CA), Diatom enerden (Sigma) or silica gel. (Diagen, DE 41 39 664 AI).
  • the patent EP 1135479 discloses that so-called for the adsorption of nucleic acids to the known and used in the art silicate materials Antichaotropic salts can be used very efficiently and successfully as a component of lysis / binding buffer systems.
  • the advantage of this method is that by avoiding the use of chaotropic salts, a significantly lower health risk emanates from the extraction systems.
  • high salt concentrations > 1.5 M
  • lysing buffers used contain salt concentrations between 1.5M-3M.
  • lysis buffers identified in the patent always contain salt concentrations of 4 M-8 M; in particular, the salts used are guanidine hydrochloride, guanidine thiocyanate or potassium iodide. It is known that these salts realize a lysis of the starting material as well as a potent inactivation of nucleolytic enzymes. After lysis of the starting material then the addition of an alcohol. The patent discloses that the addition of the alcoholic components to the high salt lysis buffer mediates a particularly good efficiency of the binding of the nucleic acids to the silicate filter materials used.
  • the lysis of the virus is carried out by means of a lysis buffer.
  • the lysis buffer is then placed on a filter column and the nucleic acid bound in a conventional manner to the filter column, washed and subsequently isolated the viral nucleic acid. Again, this process is extremely expensive and not robust for field use.
  • the object of the present invention was to eliminate the disadvantages of the solutions described in the prior art.
  • the present invention solves the problem in a surprising manner.
  • these beads have no antibodies on the surface, they surprisingly highly efficiently bind cells, bacteria and viruses, but no proteins. It turns out that the incubation of a corresponding bead solution with a cell suspension of NIH 3T3 cells results in all cells of the suspension adsorbing to the beads. This observation could also be observed on the nonspecific adsorption of bacteria or viruses.
  • viruses, bacteria or cells, etc. of a sample nonspecific adsorb to magnetic or paramagnetic materials and
  • the process according to the invention combines for the first time the adsorption of , viruses or cells etc. of a sample to a carrier material with the subsequent isolation and purification of nucleic acids using the same carrier material.
  • the method can be carried out as a “single-tube process” or else in the form of an automated "walk-away method".
  • the carrier material used is inexpensive, since it is not antibody-coupled beads. In addition, the beads are stable even without cooling and thus suitable for mobile field use.
  • the method with respect to the volume of the sample and also not with respect to the nature of the sample.
  • the last point has a decisive advantage, because it also allows samples to be processed efficiently, which can only be processed with the known methods of nucleic acid isolation consuming, especially samples with high inhibitory potential.
  • the advantage is based on the method according to the invention.
  • the cells, bacteria or viruses are adsorbed from a sample to the beads. Since proteins are not adsorbed to the beads, this is at the same time a separation of any existing free (outside of cells) existing proteins.
  • the sample can be discarded and in this context, the inhibitory matrix components.
  • the cells, bacteria or viruses etc. on the beads can now be washed with water. For the subsequent nucleic acid isolation then you have clean cells, bacteria or viruses etc .. This then simplifies the process of the actual nucleic acid isolation enormously, since expensive washing steps can be omitted.
  • the process sequence is simple, extremely fast and extremely efficient and, according to the invention, combines an enrichment of targets with the subsequent extraction of the nucleic acid.
  • the method according to the invention differs from the previously customary methods for isolating nucleic acids, since cells, bacteria, viruses etc. of a sample are first brought into contact with a carrier material.
  • previous methods always start with the lysis of the sample (cells, bacteria, viruses, etc.) and then bring the released nucleic acids into contact with a carrier material (optionally after addition of a binding buffer).
  • the method of the invention uses such a simple means as magnetic or paramagnetic materials, which are commonly used for the isolation of nucleic acids, but not for the adsorption of cells, Bacteria, viruses etc ..
  • nucleic acids For the lysis / binding of nucleic acids, in principle, all known reagents can be used, which are used for the hitherto known methods of isolating nucleic acids.
  • the method according to the invention can be used universally for the isolation of nucleic acids from liquid samples or from solid samples which have been converted into a liquid state.
  • a particular advantage of the method makes it possible that a higher diagnostic sensitivity is achievable via the use of large sample volumes. This is important, inter alia, in food diagnostics.
  • the detection of food pathogens by culturing the original sample in a Stomacher bag and a specific culture medium. Pathogen cultivation takes several hours to days. Thereafter, 1 ml of the culture is used for the nucleic acid isolation and subsequent PCR. Thus, the time until the presence of the diagnostic result is extremely long.
  • the cultivation time can be significantly reduced since, instead of the usual 1 ml sample, a much larger sample volume is used and thus the sensitivity is increased.
  • the nucleic acid is isolated and can be used in diagnostic PCR. The result is much earlier.
  • This example exemplifies the potential of this new method.
  • the invention will be explained in more detail with reference to embodiments. The embodiments do not represent a limitation of the invention.
  • Embodiments Embodiment 1 Embodiments Embodiment 1
  • the supernatant was assayed for the presence of non-adsorbed cells (isolation of the nucleic acid by a standard procedure from the supernatant, after centrifugation to pellet any cells that may be present).
  • the beads were subsequently incubated with a high salt buffer (4M guanidine thiocyanate) for cell lysis for 10 minutes at room temperature and with continuous shaking. Subsequently, the addition of isopropanol (same volume as the lysis buffer used). The batch was briefly mixed by means of a pipette and incubated for 2 min. This step serves to attach the liberated after lysis nucleic acids to the beads. Subsequently, the beads were separated by means of a magnet and the supernatant discarded. The beads were then washed twice with 80% ethanol and finally the residual ethanol was removed at 70 ° C. for 3 min.
  • a high salt buffer 4M guanidine thiocyanate
  • the beads were resuspended and incubated for 3 min at 70 ° C. Finally, the beads were separated by means of a magnet and the supernatant was transferred to a new vessel. To analyze the isolation of the nucleic acids, the DNA was applied to an agarose gel. As a control, the corresponding supernatants were used after adsorption of the cells to the beads. These supernatants were also used for DNA isolation by a standard procedure. It can be seen that with the method according to the invention the cells were first adsorbed to the beads efficiently and Subsequently, the DNA could be isolated from the cells by means of the same beads. The corresponding supernatants contained no DNA.
  • a low salt buffer 10 mM Tris HCl
  • Figure 1 shows the analysis of the isolated nucleic acids on an agarose gel.
  • the individual samples in FIG. 1 show:
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the aim of the investigation was to check whether unfunctionalized iron oxide particles for the adsorption of cells and subsequent isolation of DNA can be used. Iron oxide beads without functionalization and OH-functionalized iron oxide beads were used.
  • the supernatant was assayed for the presence of non-adsorbed cells (isolation of the nucleic acid by a standard procedure from the supernatant, after centrifugation to pellet any cells that may be present).
  • the beads were subsequently incubated with a high salt buffer (4M guanidine thiocyanate) for cell lysis for 10 minutes at room temperature and with continuous shaking. Subsequently, the addition of isopropanol (same volume as the lysis buffer used). The batch was briefly mixed by means of a pipette and incubated for 2 min. This step serves to attach the liberated after lysis nucleic acids to the beads.
  • the beads were separated by means of a magnet and the supernatant discarded. The beads were then washed twice with 80% ethanol and finally the residual ethanol was removed at 70 ° C for 3 min. After addition of a low salt buffer (10 mM Tris HCl), the beads were resuspended and incubated for 3 min at 70 ° C. Finally, the beads were separated by means of a magnet and the supernatant was transferred to a new vessel. To analyze the isolation of the nucleic acids, the DNA was applied to an agarose gel. As a control, the corresponding supernatants were used after adsorption of the cells to the beads. These supernatants were also used for DNA isolation by a standard procedure. It can be seen that with the method according to the invention both with functionalized beads and with unfunctionalized beads, the cells were first adsorbed efficiently to the beads and subsequently the DNA could be isolated from the cells by means of the same beads.
  • FIG. 2 shows the analysis of the isolated nucleic acids on an agarose gel.
  • the individual samples in FIG. 2 show:
  • the aim of the investigation was to check whether the adsorption of the cells to the beads is possible efficiently even in a very short time.
  • Each 2.5 ⁇ 10 6 NIH 3T3 cells were resuspended in 1 ml of water.
  • the supernatant was assayed for the presence of non-adsorbed cells (isolation of the nucleic acid by a standard procedure from the supernatant, after centrifugation to pellet any cells that may be present).
  • the beads were subsequently incubated with a high salt buffer (4M guanidine thiocyanate) for cell lysis for 10 minutes at room temperature and continuous shaking. Subsequently, the addition of isopropanol (same volume as the lysis buffer used). The batch was briefly mixed by means of a pipette and incubated for 2 min. This step serves to attach the liberated after lysis nucleic acids to the beads. Subsequently, the beads were separated by means of a magnet and the supernatant discarded. The beads were then washed twice with 80% ethanol and finally the residual ethanol was removed at 70 ° C for 3 min.
  • a high salt buffer 4M guanidine thiocyanate
  • the beads were resuspended and incubated for 3 min at 70 ° C. Finally, the beads were separated by means of a magnet and the supernatant was transferred to a new vessel. To analyze the isolation of the nucleic acids, the DNA was applied to an agarose gel. As a control, the corresponding supernatants were used after adsorption of the cells to the beads. These supernatants were also used for DNA isolation by a standard procedure. It turns out that with the method according to the invention the adsorption of the cells to the beads is possible even with very short incubation times. The corresponding supernatants contained no DNA.
  • FIG. 3 shows the analysis of the isolated nucleic acids on an agarose gel.
  • the individual samples in FIG. 3 show:
  • the beads were subsequently treated with solutions from a commercial kit for the isolation of nucleic acids (innuPREP DNA Mini Kit, Analytik Jena AG).
  • the beads were mixed with 300 ⁇ of a lysis buffer (Lysis Solution TLS) and 25 ⁇ proteinase K (20 mg / ml) and resuspended. Then incubation was carried out at 70 ° C for 15 min. After lysis, 300 ⁇ l of a binding buffer (Binding Solution TBS) were added. The sample was mixed thoroughly and incubated for 2 min at room temperature. This step serves to attach the released after lysis bacterial nucleic acids to the beads. Subsequently, the beads were separated by means of a magnet and the supernatant discarded.
  • the beads were then washed twice with Washing Solution MS and final at 70 ° C removed the remaining ethanol for 3 min. After addition of a low-salt buffer (elution buffer), the beads were resuspended and incubated for 3 min at 70 ° C. Finally, the beads were separated by means of a magnet and the supernatant was transferred to a new vessel.
  • Salmonella could be adsorbed to the beads used with the method according to the invention and could subsequently be isolated from the Salmonella DNA by means of the same beads and detected. It is important that the method is also suitable for enriching bacteria from large-volume samples (10 ml) and subsequently isolating nucleic acids. This is crucial if greater sensitivity is only possible over a larger sample volume.
  • FIG. 4 shows the detection of the enriched salmonella by means of the rapidSTRIPE Salmonella assay on lateral flow test strips.

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Abstract

L'invention concerne un procédé consistant à enrichir des bactéries, des cellules ou des virus provenant d'échantillons biologiques, caractérisé en ce que l'échantillon est ajouté à un échantillon liquide et mélangé à des particules magnétiques, puis ces particules magnétiques sont éliminées de l'échantillon liquide avec les bactéries, les cellules ou les virus adsorbés. L'invention concerne également un procédé consistant à isoler ensuite des acides nucléiques présents dans les bactéries, les cellules ou les virus enrichis provenant d'échantillons biologiques, caractérisé en ce que les bactéries, les cellules ou les virus enrichis sont lysés selon des procédés connus et que les acides nucléiques sont isolés selon un procédé connu.
PCT/EP2011/062161 2010-07-15 2011-07-15 Procédé pour enrichir des bactéries, des virus et des cellules, et pour isoler ensuite leurs acides nucléiques WO2012007581A1 (fr)

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DE102010031401.3 2010-07-15
DE201010031401 DE102010031401A1 (de) 2010-07-15 2010-07-15 Verfahren zur Anreicherung von Bakterien, Viren sowie Zellen und zur nachfolgenden Nukleinsäureisolierung

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014029792A1 (fr) 2012-08-21 2014-02-27 Qiagen Gmbh Stabilisation de particules virales et procédé d'isolement d'acides nucléiques viraux
CN110167952A (zh) * 2016-07-25 2019-08-23 Aj耶拿检疫有限公司 用于富集生物分子并且用于从生物样品中去除这些生物分子的方法
EP3286325B1 (fr) * 2015-04-23 2022-07-13 IST Innuscreen GmbH Procédé et kit d'essai pour l'isolation rapide d'acides nucléiques au moyen de surfaces rugueuses

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102017204195A1 (de) * 2017-03-14 2018-09-20 Robert Bosch Gmbh Verfahren und mikrofluidische Vorrichtung zur Prozessierung von Viren und Bakterien einer Probe
DE102017204267B4 (de) 2017-03-14 2021-05-27 Aj Innuscreen Gmbh Verfahren zur anreicherung von zellen aus einer probe und der nachfolgenden nukleinsäureisolierung aus diesen zellen
DE102018132710A1 (de) 2018-12-18 2020-06-18 Analytik Jena Ag Filtrierverfahren geeignet zur Isolierung und/oder Quantifizierung zumindest einer zu untersuchenden Substanz aus einer Probe
DE102019118332B4 (de) * 2019-07-07 2022-04-07 Ist Innuscreen Gmbh Verfahren und testkit zur bisulfitmodifizierung von dna

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US5234809A (en) 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
DE4307262A1 (de) * 1993-03-02 1994-09-08 Christian Bergemann Magnetisches polymeres Siliciumdioxid
DE4321904A1 (de) 1993-07-01 1995-01-12 Diagen Inst Molekularbio Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen
WO1995034569A1 (fr) 1994-06-14 1995-12-21 Invitek Gmbh Procede universel d'isolement et de purification d'acides nucleiques a partir de quantites extremement reduites de differents materiaux de depart fortement contamines
WO1998051693A1 (fr) * 1997-05-13 1998-11-19 Genpoint As Isolation en phase solide d'acides nucleiques
EP1135479A1 (fr) 1998-12-04 2001-09-26 Invitek GmbH Formulation et technique permettant d'isoler des acides nucleiques a partir d'un materiel de depart complexe, et analyse genique complexe leur faisant suite
EP1621618A1 (fr) * 1999-05-14 2006-02-01 Promega Corporation Concentration de cellules et élimination de lysats au moyen de particules paramagnétiques
EP1655366A2 (fr) * 2004-11-03 2006-05-10 Samsung Electronics Co., Ltd. Appareil et méthode pour la purification d'acides nucléiques par séparation de phases utilisant un laser et des billes
EP1944368A1 (fr) * 2007-01-15 2008-07-16 Konica Minolta Medical & Graphic, Inc. Procédé d'isolation d'acides nucléiques par chauffage sur support magnétique

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CN1230531C (zh) * 2002-12-09 2005-12-07 清华大学 从样品中分离细胞粒子的方法

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US5234809A (en) 1989-03-23 1993-08-10 Akzo N.V. Process for isolating nucleic acid
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DE4321904A1 (de) 1993-07-01 1995-01-12 Diagen Inst Molekularbio Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen
WO1995034569A1 (fr) 1994-06-14 1995-12-21 Invitek Gmbh Procede universel d'isolement et de purification d'acides nucleiques a partir de quantites extremement reduites de differents materiaux de depart fortement contamines
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EP1621618A1 (fr) * 1999-05-14 2006-02-01 Promega Corporation Concentration de cellules et élimination de lysats au moyen de particules paramagnétiques
EP1655366A2 (fr) * 2004-11-03 2006-05-10 Samsung Electronics Co., Ltd. Appareil et méthode pour la purification d'acides nucléiques par séparation de phases utilisant un laser et des billes
EP1944368A1 (fr) * 2007-01-15 2008-07-16 Konica Minolta Medical & Graphic, Inc. Procédé d'isolation d'acides nucléiques par chauffage sur support magnétique

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014029792A1 (fr) 2012-08-21 2014-02-27 Qiagen Gmbh Stabilisation de particules virales et procédé d'isolement d'acides nucléiques viraux
US10233508B2 (en) 2012-08-21 2019-03-19 Qiagen Gmbh Virus particle stabilisation and method for isolating viral nucleic acids
EP3286325B1 (fr) * 2015-04-23 2022-07-13 IST Innuscreen GmbH Procédé et kit d'essai pour l'isolation rapide d'acides nucléiques au moyen de surfaces rugueuses
CN110167952A (zh) * 2016-07-25 2019-08-23 Aj耶拿检疫有限公司 用于富集生物分子并且用于从生物样品中去除这些生物分子的方法

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