EP1981975A2 - Composition for stabilizing biological samples and method of stabilization - Google Patents
Composition for stabilizing biological samples and method of stabilizationInfo
- Publication number
- EP1981975A2 EP1981975A2 EP07703775A EP07703775A EP1981975A2 EP 1981975 A2 EP1981975 A2 EP 1981975A2 EP 07703775 A EP07703775 A EP 07703775A EP 07703775 A EP07703775 A EP 07703775A EP 1981975 A2 EP1981975 A2 EP 1981975A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- composition according
- agent
- composition
- solution
- stabilization
- 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.)
- Withdrawn
Links
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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
-
- 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
Definitions
- composition for the stabilization of biological samples and stabilization process Composition for the stabilization of biological samples and stabilization process
- the present invention relates to a new composition for the stabilization of biological samples, in particular nucleic acids, allowing their conservation for several months without necessarily having to keep them at low temperature.
- the most used methods in biology are those for amplifying the information present in nucleic acid sequences, such as techniques derived from PCR. Quantitation and detection of the information present in the nucleic acid sequences have multiple applications, for example the identification of biological markers with potential applications in terms of diagnosis, prognosis or patient follow-up.
- a good procedure for extraction and purification of nucleic acids from biological samples is essential to obtain the information resulting from molecular biology experiments.
- the procedures for analyzing biological samples generally comprise a set of steps for obtaining pure nucleic acids, and then proceed to molecular biology analyzes: Step 1: Lysis of the cells.
- Step 2 Degradation of proteins (inactivation, proteolysis). - Step 3: Elimination of proteins and contaminants (cell lysate ). Step 4: Recovery of the DNA or RNA. Step 5: Storage (if necessary). Step 6: Molecular biology experiment (PCR, quantitative PCR ).
- the first four steps are performed sequentially (different manipulations and different reagents). These four steps must also be performed in the same time space because of the impossibility of stopping the process, at the risk of changing the reaction efficiency (which would not then allow to have a standardized nucleic acid manipulation between each treated sample).
- Step 1 These kits and procedures allow a short stabilization of a few hours at room temperature, to several days for storage at controlled temperatures. Such kits and procedures are described in the documents accompanying the marketing of these products, such as the Stratagene SideStep TM kits, the company's RNAlater TM
- the present invention thus relates to a new ready-to-use composition, making it possible to implement a simplified method for stabilizing and storing biological samples.
- it makes it possible to reduce steps 1 and 2 described above in a single step.
- it is possible to preserve the samples thus treated over a long period of time.
- composition according to the invention also brings a significant economic advantage, in particular because the storage can be carried out at ambient temperature.
- the present invention thus relates to a composition for the stabilization of biological samples, this composition comprising
- stabilizing composition is preferably meant according to the invention a composition comprising the above constituents, and if necessary below, before incorporating the biological sample to be stabilized.
- composition according to the invention does not comprise said biological sample.
- composition according to the invention may be stored in solid form or in aqueous solution. It will be used in aqueous form for the stabilization of biological samples.
- the pH preferably of this solution will be between 4 and 11. Depending on the deactivating agent selected, the preferred pH may vary. Thus, for a detergent, the pH will more preferably be between 4 and 5, even more preferably close to 4.5.
- a chelating agent it will more preferably be between 7 and 11, even more preferably between 8 and 10, advantageously close to 9.
- the pH may be adjusted to the preferred values by the use of a base or an appropriate acid.
- the composition in the form of a solution may also comprise a buffer or an acid or a base (D) so as to adjust and / or stabilize the pH to the desired value.
- solution is meant according to the invention an aqueous composition comprising, in addition to water, the constituents (A) to (C) and, where appropriate, the buffer (D) in the form of a true solution, or else a suspension, or an emulsion, or any other physical form resulting from the mixing of the various constituents in the water.
- the composition according to the invention when it is a ready-to-use stabilizing solution, it comprises, in addition to water, the components (A) to (D) in the following proportions: deactivating agent (A): from 1/4 to 1/6 by volume of the total volume of the solution, preferably 1/5 by volume for a detergent and between 175 and 225 mM for a chelating agent.
- deactivating agent (A) from 1/4 to 1/6 by volume of the total volume of the solution, preferably 1/5 by volume for a detergent and between 175 and 225 mM for a chelating agent.
- chaotropic agent or denaturing agent B): from 5 to 5 M (mole / l), preferably 6 M.
- peptidase (C) from 1.5 to 2 mg / ml, preferably 1.8 mg / ml, and, where appropriate buffer (D): to adjust the pH between 4 and 1.
- the proportions of the components (A) to (D) are suitable to allow the preparation of a solution as defined above.
- the stabilizing solution according to the invention is advantageously prepared by adding to a given quantity of water, the constituents (A) and (B), then to adjust the pH by adding the buffer (D) before adding the peptidase (C).
- the composition may be prepared extemporaneously, each of its constituents being mixed for the preparation of the aqueous composition.
- pre-mixtures of constituents may be prepared and stored, for example the deactivation agent (A), the chaotropic agent (B) and / or the denaturing agent (B) on the one hand, and peptidase (C) on the other hand.
- the premix comprising the deactivating agent (A), the chaotropic agent (B) and / or if appropriate, the denaturing agent (B) and / or the buffer (B), is also called a mixture 1 in the present patent application.
- premixes can be in liquid form, a premix solution, or in solid form.
- the solid forms of premixes such as the composition of according to the invention, can be prepared by adding the various pure components in appropriate proportions for the preparation of the stabilizing solution, or by preparing a solution comprising the various constituents in the appropriate proportions, and then removing the water in a suitable manner. Substantial by any suitable means, for example by usual lyophilization techniques.
- the peptidase (C) also lyophilized, can be added.
- This freeze-dried stabilization premix can be stored at controlled temperature, around 4 ° C.
- the invention therefore also relates to a stabilization kit for biological samples comprising the constituents of the composition, isolated or premixed in proportions suitable for producing a solution according to the invention, ready for use.
- the kit may further comprise a container, for example a sample tube, a plate or a laboratory-on-a-chip, in which the biological sample mixed with the solution according to the invention will be treated and then preserved.
- the "lab on chip” or “Lab on Chip” is advantageously a consumable on a “chip” type support, said “chip” comprising freeze-dried reagents distributed on its surface or in appropriate volumes formed in said support, the mixture being extemporaneously with the passage of the sample to be analyzed.
- the appropriate volumes or freeze-dried reagents are distributed in a standardized manner on the support, plate or "chip".
- the amounts of each constituent in the premixes and in the kit according to the invention are so-called “ready-to-use” quantities, that is to say appropriate to obtain a stabilizing solution comprising the preferred proportions of each component discussed above, without having to perform new assays.
- detergent is meant according to the invention a compound having detergent or chelating properties, that is to say selected from detergents or chelating agents.
- detergent is meant according to the invention any compound for which the hydrophilic / hydrophobic balance is substantially balanced and which allows the lysis of the bi-lipidic membrane of the cells.
- Such detergents may be neutral, anionic, cationic or zwitterionic and are well known to those skilled in the art.
- the following compounds are particularly suitable:
- the detergent is a nonionic detergent, considered a non-denaturing average activity detergent, often used in biochemical applications to solubilize proteins, lyse cells, and not necessarily having an antimicrobial property.
- the nonionic detergent is a detergent of the "Triton X" series, produced by polymerization of octylphenol with ethylene oxide, more preferably Triton X-100 (Triton X-100 is a product of Union Carbide Triton is a registered trademark of Rohm and Haas Company, Philadelphia, PA, USA).
- Triton X-100 is a product of Union Carbide Triton is a registered trademark of Rohm and Haas Company, Philadelphia, PA, USA.
- nonionic detergents such as Igepal and Nonidet may advantageously be employed in the composition according to the invention.
- chelating agent any hydrocarbon molecule having the ability to combine with ions to form complexes.
- Chelating agents are well known to those skilled in the art and generally include amino acids linked by non-peptide bonds. It is in particular polyamines, preferably diamines, aliphatic, each amino group being substituted by at least one hydrocycarbonyl-alkyl radical.
- the preferred chelating agents according to the invention may be represented by the general formula (i):
- n independently represents an integer from 1 to 3.
- Compound B is either a "chaotropic agent” or a “denaturing agent” or a mixture of both.
- the term "chaotropic agent” is intended to mean an agent which makes it possible to separate / isolate the nucleic acids from their protein part which makes it possible to protect the nucleic acids from proteins which can damage them, in particular DNAses and RNases.
- chaotropic agents are well known to those skilled in the art, and more particularly the guanidine salts (Cox, RA, The Use of Guanidinium Chloride in the Isolation of Nucleic Acids, Methods in Enzymology, 12B, 120-129 (1968) Data for Biochemical Research, 3rd ed., Dawson, RMC, et al, Oxford University Press (New York, NY: 1986), pp.
- denaturing agent is understood according to the invention an agent leading to protein denaturation, more particularly resulting from the modification (disruption) of the native conformation (biologically active conformation) of a protein, without rupture of peptide bond (hydrolysis) by modification of one or more physicochemical parameters of its environment It may be said that the denaturing agents "unwind" the globular proteins The denaturing agent (D) thus makes it possible to alter the activity of the enzymes present in the biological sample, as DNAses or RNAses that would be likely to degrade nucleic acids in biological samples.
- proteases are proteases or proteolytic enzymes, enzymes that cleave the peptide bonds of proteins. This process is called proteolytic cleavage.
- proteases are well known to those skilled in the art (http://www.expasy.ch/cgi-bin/lists7peptidas.txt or http://merops.sanger.ac.uk/).
- the peptidase is Proteinase K. Proteinase K is a stable and highly reactive plant serine protease (Ebling,
- Proteinase K has no hydrolysis effect on nucleic acids (Sweeney, PJ and Walker, J.M., Burrell, M. M., Enzymes of Molecular Biology). It hydrolyzes proteins of all origins in a few hours with a preference for peptide bonds located after hydrophobic amino acids (eg leucine). Proteinase K is used in nucleic acid preparation and purification techniques to digest tissues / cells, membranes, etc.
- Buffers (D) used in the field of biology are well known to those skilled in the art. They preferably have the following characteristics: soluble in water, without interference with biological processes and preferably non-toxic. We can mention the following buffers:
- pH of the prepared solution can be adjusted, in addition to the use of the buffer, with the addition of an appropriate amount of acid, such as hydrochloric acid.
- the buffer according to the invention is a Tris HCl buffer (mixture tris (hydroxymethyl) aminomethane and HCl), or a buffer based on citric acid or acetic acid.
- the water used for its preparation will preferably be treated water so as to eliminate any biological contamination and more preferably biological, organic and mineral, more particularly distilled or deionized water, as recommended by good laboratory practice.
- the water used for the preparation of the liquid sample may also be the water contained in the biological sample taken, for example when this sample is a blood sample.
- the stabilizing composition comprises
- composition according to the invention corresponding to this first embodiment is an aqueous stabilization solution comprising, in addition to water, the following constituents:
- the pH is usually adjusted by adding the buffer and optionally the acid before adding the peptidase to the premix.
- the invention therefore also relates to a premix, or mixture 1, for producing a solution comprising: Triton X-100 1/5 of the final volume (v / v) guanidine-HCl 5.9M 10 mM Urea
- the mixture 1 is a lyophilizate of a solution comprising the components (A), (B), (D) in the proportions above.
- the stabilization composition comprises:
- composition according to this second mode may comprise an appropriate amount of alkali metal salt, in particular sodium chloride.
- the pH is preferably adjusted to between 7 and 11, more preferably between 8 and 10, and even more preferentially around 9 by the addition of an organic or inorganic base, preferably sodium hydroxide.
- the pH can also be adjusted by adding an appropriate buffer defined above.
- the composition according to the invention corresponding to this second embodiment is a solution which comprises, in addition to water, the following elements:
- the pH is generally adjusted by adding the buffer and optionally the base before adding the peptidase to the premix.
- the invention therefore also relates to a premix, or mixture 1, for producing a solution comprising:
- the mixture 1 is a lyophilizate of a solution comprising the components (A), (B), (D) and (E) in the proportions above.
- the invention also relates to a stabilization kit comprising, on the one hand, the mixture 1 defined above, and on the other hand an appropriate quantity of proteinase K, allowing the production of a stabilization solution comprising 1.8 mg / ml of this peptidase.
- the techniques for taking biological samples, processing, recovering DNA or RNA and any subsequent use of the stabilized samples remain unchanged compared to prior techniques.
- the stabilization solution according to the invention makes it possible to simplify the process by combining the lysis of the cells and the degradation of the proteins in a single step. It allows above all to keep the samples thus treated for several months without the need to control the storage temperature.
- the samples may be stored at ambient temperature, preferably below 30 ° C., more preferably below 25 ° C.
- the samples treated in the stabilizing solution according to the invention can be stored for more than 12 months at 25 ° C. Of course, they can be stored in a temperature controlled environment of about 4 ° C, allowing even longer storage, until at least 18 months.
- the invention therefore also relates to a method for preparing stabilized biological samples, said method comprising the following steps: mixing a stabilized biological sample with a stabilization composition according to the invention, incubating the mixture to allow cell lysis and degradation of the proteins, and storage of the stabilized mixture obtained previously.
- the incubation conditions are conditions common to those skilled in the art, advantageously at a temperature greater than 30 ° C., more preferably greater than 30 ° C.
- the duration of the incubation step will depend on the incubation temperature. For a temperature of 70 ° C., the incubation will be carried out between
- the storage is carried out at room temperature for a duration of more than 2 days.
- the stabilization solution according to the invention it is possible to store the biological samples at room temperature for more than 3, 4 or 5 days, and according to the uses more than one week, or even more than 2, 3 or 4 weeks, or more from one month to more than one year, without observing any substantial degradation of the DNA contained in the biological sample taken.
- the stabilizing solution and the method according to the invention are particularly suitable for the collection and preservation of samples containing genomic DNA.
- the biological sample used in the method according to the invention comprises any biological sample taken from an organism, living or not, especially from fresh whole blood, frozen blood, bone marrow tissues, oral cells, cell cultures. , liquid samples etc
- the incubation step is carried out when the stabilizing composition according to the invention comprising the sample is in a liquid form.
- the composition according to the invention used in the process is an aqueous composition.
- the composition is in a solid form, for example in freeze-dried form, to which a biological sample in liquid form, for example blood or any other liquid sample, is added. water being optionally added to obtain the desired volume.
- the liquid biological sample is added to a premix according to the invention, and the volume is then completed, if necessary, with water, and the peptidase (C) is then added. .
- the stabilization method can be implemented in a "high speed" type application.
- the "Mix 1" solution in liquid form can be deposited at the bottom of the plate (96/384 wells or other standardized formats used for the collection of biological samples) and then the proteinase (C) is added.
- the biological sample can then be added in a systematic and standardized way.
- the stabilized samples according to the invention can then be processed in the usual manner to extract the DNA, in particular the gDNA or RNA contained in the biological sample according to different methods: extraction kit using a silica column. extraction kit using an affinity column (Anion-exchange methods). extraction kit using an extraction method with organic solvents. extraction kit using a cesium chloride density gradient method, extraction kit using a precipitation method (Desalting method). extraction kit using magnetic beads, in particular coated with silica.
- Figure 1 shows the general scheme for the preparation of samples stabilized with the method and the solution according to the invention.
- FIGS 2 to 4 show the results of the conservation tests of the stabilized biological samples.
- An Eppendorf tube (a tube for a sample).
- Figure 4 shows the 180-day photo-radiograms of the high-molecular-weight controls and control gene amplification for samples stored at 4 ° C ( Figure 4.A), and 30 days at room temperature ( Figure 4.B).
- the control gene is the HPRT for samples stored at 40 ° C. and at room temperature. at.
- Migration on agarose gel 0.8 to 1% after purification verification of the absence of smear (which would indicate a degradation of the gDNA) and conversely, the presence of a band of very high molecular weight greater than 15 Kb (Fig. 4.1 High PM).
- b. PCR test on control gene, example HPRT, Actin ... Fig. 4.2 Control gene: shows that after stabilization and purification, the enzymatic reactions are possible.
- vs. Spectrometric analysis (Fig 2 and 3)
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0600182A FR2895993A1 (en) | 2006-01-10 | 2006-01-10 | COMPOSITION FOR STABILIZING BIOLOGICAL SAMPLES AND METHOD OF STABILIZATION |
PCT/EP2007/050228 WO2007080178A2 (en) | 2006-01-10 | 2007-01-10 | Composition for stabilizing biological samples and method of stabilization |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1981975A2 true EP1981975A2 (en) | 2008-10-22 |
Family
ID=36146943
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP07703775A Withdrawn EP1981975A2 (en) | 2006-01-10 | 2007-01-10 | Composition for stabilizing biological samples and method of stabilization |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1981975A2 (en) |
FR (1) | FR2895993A1 (en) |
WO (1) | WO2007080178A2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK2742152T3 (en) * | 2011-08-12 | 2017-07-31 | Qiagen Gmbh | PROCEDURE FOR ISOLATING NUCLEIC ACIDS |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19743518A1 (en) * | 1997-10-01 | 1999-04-15 | Roche Diagnostics Gmbh | Automated, universally applicable sample preparation method |
DE19836559A1 (en) * | 1998-08-12 | 2000-03-23 | Antigen Gmbh | Blood collection vessel |
WO2000029618A1 (en) * | 1998-11-12 | 2000-05-25 | University Of Virginia Patent Foundation | Non-invasive detection of helicobacter pylori infection |
CA2428864C (en) * | 2000-11-08 | 2011-04-12 | Becton, Dickinson And Company | Method and device for collecting and stabilizing a biological sample |
EP1524317B1 (en) * | 2003-10-13 | 2015-03-04 | Roche Diagnostics GmbH | Methods for isolating nucleic acids |
ITMI20040167A1 (en) * | 2004-02-04 | 2004-05-04 | Univ Padova | METHOD FOR SIMULTANEOUS EXTRACTION OF NUCLEIC ACIDS FROM A BIOLOGICAL SAMPLE |
WO2005093065A1 (en) * | 2004-03-16 | 2005-10-06 | Roche Diagnostics Gmbh | Improved method of isolating nucleic acids |
-
2006
- 2006-01-10 FR FR0600182A patent/FR2895993A1/en active Pending
-
2007
- 2007-01-10 EP EP07703775A patent/EP1981975A2/en not_active Withdrawn
- 2007-01-10 WO PCT/EP2007/050228 patent/WO2007080178A2/en active Application Filing
Non-Patent Citations (1)
Title |
---|
See references of WO2007080178A3 * |
Also Published As
Publication number | Publication date |
---|---|
FR2895993A1 (en) | 2007-07-13 |
WO2007080178A3 (en) | 2007-09-07 |
WO2007080178A2 (en) | 2007-07-19 |
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