EP3765635A1 - Verfahren zur extraktion von nukleinsäure - Google Patents

Verfahren zur extraktion von nukleinsäure

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Publication number
EP3765635A1
EP3765635A1 EP19717541.7A EP19717541A EP3765635A1 EP 3765635 A1 EP3765635 A1 EP 3765635A1 EP 19717541 A EP19717541 A EP 19717541A EP 3765635 A1 EP3765635 A1 EP 3765635A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acids
sample
masking
proteins
reagent
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.)
Pending
Application number
EP19717541.7A
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English (en)
French (fr)
Inventor
Alain Laurent
Arnaud Burr
Ali Laayoun
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.)
Biomerieux SA
Original Assignee
Biomerieux SA
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 Biomerieux SA filed Critical Biomerieux SA
Publication of EP3765635A1 publication Critical patent/EP3765635A1/de
Pending legal-status Critical Current

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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
    • 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 present invention relates to a method for extracting nucleic acids using a suitable solid support, for example, based on silica.
  • the method comprises a nucleic acid capture step by contacting the sample with a suitable solid support, characterized in that, prior to the capture step, said method comprises a sample treatment step with at least one reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides of the sample.
  • nucleic acids from complex biological samples (blood, tumor, food, etc.) which consists of a chemical or mechanical lysis of the cells in order to release the contents and particularly the nucleic acids. The latter are then purified selectively and then amplified if their quantity is not sufficient for direct detection.
  • nucleic acid extraction techniques use solid phases where the cells are lysed under specific reaction conditions and the released nucleic acids bind to the solid phase. It is well known in the state of the art that current nucleic acid extraction techniques very often implement solid phases which are, for example, particles coated with silica. Silica has the property of reversibly adsorbing nucleic acids under certain salt concentration and pH conditions, making it a very suitable material for this purpose. These techniques are described for example in "Rapid and simple method for purification of nucleic acids. Boom, Journal of Clinical Microbiology, 1990 p495 or in US Pat. No. 5,234,809 by the same author.
  • the present disclosure is directed to providing methods for extracting nucleic acids that meet one or more of the following criteria:
  • the present disclosure relates to a method of extracting nucleic acids from a sample comprising proteins and / or polysaccharides, said method comprising a step of capturing nucleic acids by contacting the sample with a suitable solid support , characterized in that, prior to the capture step, said method comprises a step of treating the sample with at least one reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides of the sample .
  • the function masking reagent amines is selected from acylating or alkylating agents.
  • the masking reagent is chosen from the acylating agents of formula (I) below:
  • R is an organyl, organyloxy or organylamino group
  • LG is a leaving group selected from the group consisting of halogens, organyloxy groups and organylamino groups.
  • the masking reagent may be an acylating agent selected from the group consisting of activated esters, acid halides, chloroformates, anhydrides, activated carbonate esters, and carbonyl diimidazole.
  • the masking reagent is an anhydride selected from acetic anhydride, propanoic anhydride, isobutyric anhydride, butanoic anhydride or benzoic anhydride.
  • the masking reagent is an alkylating agent selected from the group consisting of alkyl halides, diazo compounds, and aldehydes.
  • the masking reagent is selected from amines, alcohols and thiols for masking the carboxylic acid functions of the proteins, in combination with a coupling agent.
  • a coupling agent for example R1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), di-isopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC).
  • the method for extracting nucleic acids from a biological sample comprises the following steps: a. lysis of the cells, for example by bringing the biological sample into contact with a lysis buffer,
  • washing the support with a washing buffer d. where appropriate, washing the support with a washing buffer and,
  • the solid support may consist of silica particles, in particular magnetic silica particles.
  • the biological sample is a sample of blood, plasma or serum.
  • the capture step is performed in the presence of chaotropic agents.
  • the extraction method does not include the use of proteases for protein removal.
  • no organic solvent is added before or during the nucleic acid capture step.
  • less than 50 ⁇ l, preferably less than 10 ⁇ l, and even more preferably less than 1 ⁇ l of organic solvent is added, before or during the nucleic acid capture step, per 100 ⁇ l of sample.
  • the method comprises an additional step of detecting nucleic acids of interest, in particular by amplification of the extracted nucleic acids.
  • nucleic acid extraction kits comprising at least i. a reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides as defined above,
  • a suitable solid support for example based on silica, for the extraction of the nucleic acids, iii. if necessary, a catalyst of the reaction for masking the amine and / or carboxylic acid functions using the reagent,
  • the present disclosure also relates to the use of a reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides as defined above, for the inhibition of nuclease.
  • the nuclease to be inhibited is included in a lysate obtained by lysis of a biological sample, for example, for amplification or detection of a nucleic acid in the sample.
  • extraction is meant a technique for isolating nucleic acids from any sample, for example, isolation of DNA and / or RNA from eukaryotic cells, prokaryotic, human animal, microorganism or tissue.
  • the extraction from biological sample within the meaning of the invention generally includes lysis of cells and purification of nucleic acids from the lysate.
  • the purification itself comprises the capture of the nucleic acids on a suitable solid support, preferably based on silica, the washing optionally followed by the elution of the nucleic acids.
  • the capture consists of adsorbing the nucleic acids on the solid support and the elution, when it takes place, consists of the desorption or release of the latter from the solid support.
  • sample any type of sample comprising at least nucleic acids, and agents inhibiting the extraction of nucleic acids, such as proteins and / or polysaccharides.
  • the sample may have various origins such as food, environmental, human, veterinary or cosmetic samples. All these samples, if they are not liquid, are previously treated to be in liquid form. The sample used in the extraction process is therefore in liquid form. Preferably, the sample used in the extraction process is an unbound sample.
  • Non-fixed sample means a sample that has not been processed to be preserved in its original state. Fixing a sample is a a technique well known to those skilled in the art, which may for example be carried out with an aldehyde, such as formaldehyde or glutaraldehyde.
  • Examples of food-based samples include, but are not limited to, a sample of milk products (yogurts, cheeses, etc.), meat, fish, eggs, fruit, vegetables, and beverages. (milk, fruit juice, soda, etc.). Of course, these food-based samples may also come from sauces or more elaborate dishes or unprocessed or partially processed raw materials.
  • a food sample may also be derived from a feed intended for animals, such as cakes, animal meal.
  • the sample may be of environmental origin and may consist of, for example, surface sampling, water, etc.
  • the sample may also consist of a biological sample, of human or animal origin, which may correspond to samples of biological fluid (urine, whole blood or derivatives such as serum or plasma, sputum or saliva, pus, cerebrospinal fluid, etc.), stool (eg cholera diarrhea), nose, throat, skin, wounds, organs, isolated tissues or cells, swab specimens, broncho specimens or washes -Alveolar, biopsies. This list is obviously not exhaustive.
  • biological fluid urine, whole blood or derivatives such as serum or plasma, sputum or saliva, pus, cerebrospinal fluid, etc.
  • stool eg cholera diarrhea
  • nose, throat skin, wounds, organs, isolated tissues or cells
  • swab specimens broncho specimens or washes -Alveolar, biopsies.
  • sample generally refers to a part or quantity, more particularly a small part or a small quantity, taken from one or more entities for analysis. This sample may possibly have undergone prior treatment, involving for example mixing, dilution or grinding steps, in particular if the starting entity is in the solid state.
  • the sample analyzed is, in general, capable of - or suspected of - containing at least nucleic acids representative of the presence of microorganisms, a patient's condition (for example immunosuppression, pregnancy %) or a disease to detect, characterize or follow.
  • the sample comprises isoelectric point proteins less than 8, or even less than 7.
  • the sample comprises at least one of the following proteins: human serum albumin (HSA), fibrinogen, immunoglobulins, especially IgG, and hemoglobin.
  • micro-organism By a “micro-organism” is meant all or part of a bacterium, a fungus, a yeast, a protozoan or a virus.
  • nucleic acids DNA or RNA of all types: genomic DNA, complementary DNA, messenger RNA, complementary RNA, transfer RNA, mitochondrial RNA, chloroplast DNA, ribosomal RNA, plasmid DNA, Viral DNA or RNA, microRNA, snoRNA, siRNA, RNAi, single-stranded or double-stranded form.
  • proteins any molecule comprising at least one polypeptide chain, characterized by a sequence of amino acid residues linked together by peptide bonds. This includes, in particular, peptides and polypeptides and any modified polypeptide or its derivatives resulting from post-translational or other modifications, their degradation products, in particular by enzymatic degradation, lipoproteins, etc.
  • appropriate solid support is meant any support capable of participating in the extraction of nucleic acids from a biological sample.
  • it is a support comprising or consisting of silica or one of these derivatives (silicate, glass, silica modified with organic groups, etc.), magnetic or otherwise, and capable of participating in the process.
  • extraction of nucleic acids from a biological sample may also be a carrier based on paper, cellulose, or pure magnetite, or other polymers known for their uses in the extraction of nucleic acids. It can be at least a plane support, a hollow support, a slab, a needle, a membrane, a plate, a sheet, a cone, a tube, fibers, a marble or a particle.
  • Solid support is preferably a ball or particle or membrane.
  • the solid support is preferably magnetic.
  • the method comprises the following steps: a) lysis of the cells, for example by bringing the biological sample into contact with a lysis buffer,
  • step b) is directly followed by step c), that is to say that there is no intermediate step, for example no step of deprotection of the masked functions between steps b) and c).
  • the nucleic acid extraction process does not include a deprotection step of the amine and / or masked carboxylic acid functions.
  • the present disclosure also relates to the use of a reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides in a method for extracting nucleic acids from a biological sample using a solid support. appropriate.
  • the lysis step involves breaking the sample cells (cell walls and membranes) to release the nucleic acids.
  • the cells are chemically lysed by contacting the sample with a lysis buffer.
  • the lysis buffer must be both effective in breaking the cell membranes and soft enough not to degrade the nucleic acids.
  • the lysis buffer may comprise, for example, a detergent and optionally a chelating agent.
  • the pH is generally maintained between 4 and 8, for example between 6 and 8, using a suitable buffer, for example Tris HCl, optionally in concentrations of between 10 and 100 mM.
  • the detergent may be selected from Tween, Newts, SDS and other detergents commonly used at concentrations of between 0.05 and 20%.
  • the lysis buffer contains reagents for the inactivation of nucleases, and / or the removal of proteins, for example proteases such as proteinase K.
  • Other enzymes can also be used as lytic enzymes (hydrolase, zymolase %) to digest the wall of yeasts and fungi.
  • the lysis buffer does not include reagents for the removal of proteins and especially proteases.
  • the masking reagent of the amine and / or carboxylic functions used in the extraction process according to the present disclosure advantageously also allows an inhibition of the enzyme activity of the nucleases possibly released into the lysate.
  • the lysis buffer also comprises chaotropic agents.
  • Chaotropic agents interfere with weak (non-covalent) intramolecular interactions, such as hydrogen bonds, van der Waals forces, and hydrophobic forces.
  • chaotropic agents mention may be made of urea, guanidine salts such as guanidinium chloride or thiocyanate and lithium perchlorate. They are generally used in concentrations ranging from 1 to 6 M, in particular for GuSCN and GuHCl.
  • the lysis buffer does not include chaotropic agents.
  • Chelating agents such as EDTA or similar compounds, for example between 5 and 50 mM and / or reducing compounds such as DTT (dithioerythritol) or TCEP (tri carboxyethylphosphine), or beta-mercaptoethanol at concentrations of, for example, 0.5 to 100 mM.
  • the lysis buffer can also comprise organic solvents such as alcohols (ethanol, isopropanol, etc.). In a preferred embodiment however, the lysis buffer does not include organic solvents.
  • the chemical characteristics of a sample must be taken into account when optimizing the lysis buffer.
  • the acidity of a sample eg, some soil samples
  • the sample is contacted with the lysis buffer for a time sufficient to allow lysis of the cells without degrading the nucleic acids, for example between 0 and 15 min.
  • An essential characteristic of the nucleic acid extraction process concerns the step of treating the lysate using a reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides of the lysate. 'sample.
  • This treatment step can be carried out by adding the masking reagent to the lysate after the lysis step.
  • the masking reagent is included directly in the lysis buffer, the lysis and treatment of the sample being concomitant.
  • the objective is to neutralize the charged functions of the proteins and / or polysaccharides that may affect the extraction yield using a solid support, preferably based on silica.
  • the inventors have demonstrated the impact of bio-molecules on the extraction yield, and in particular proteins and / or polysaccharides of biological samples in extraction processes using supports.
  • solids such as particles coated with silica.
  • the proteins and / or polysaccharides may in particular interact with the silica beads and / or the nucleic acids via their charged functions, and in particular their amine and / or carboxylic acid functions.
  • masking reagent any chemical compound capable of reacting (preferably irreversibly) with at least certain amine and / or acid functional groups.
  • carboxylic proteins and / or polysaccharides present in a lysate of a biological sample so as to change the properties of polarity, isoelectric point, and / or charge of these proteins and / or polysaccharides.
  • the masking reagent makes it possible to mask the charged functions, and especially the amine and / or carboxylic acid functions of the proteins and / or polysaccharides.
  • the nucleic acids are not modified by the masking reagent.
  • the reaction conditions, and in particular the concentration of masking reagent can be chosen so that the nucleic acids are not modified during the masking step.
  • the concentration of masking reagent or coupling agent, when present is between 0.01 M and 1.8 M, for example between 0.1 M and 1.0 M for example between 0.2 and 0.6 M.
  • the masked amine and / or carboxylic acid functions of the modified proteins and / or polysaccharides remain masked throughout the duration of the nucleic acid extraction process according to the present disclosure.
  • the nucleic acid extraction process does not include a deprotection step of the amine and / or masked carboxylic acid functions.
  • the masking reagent is chosen from acylating or alkylating agents, making it possible to mask the amine functions of the proteins or polysaccharides.
  • the acylating or alkylating agent must be able to mask the amine functions of the proteins or polysaccharides in an aqueous medium.
  • an acylating agent that can be used as a masking reagent in the nucleic acid extraction process has the following formula (I):
  • R is an organyl, organyloxy or organylamino group
  • LG is a departing group.
  • LG is meant an atom or a group which, during the acylation reaction, detaches from the carbon atom to which it is attached.
  • LG is a leaving group selected from the group consisting of halogens, organyloxy and organylamino groups.
  • the masking reagent may be an acylating agent selected from the group consisting of activated esters, acid halides, chloroformates, anhydrides, activated carbonate esters and carbonyl diimidazole.
  • activated esters mention may be made of tetra or pentafluorophenyl acetate, nitrophenyl acetate, pentafluorophenyl trifluoroacetate, and N-hydroxysuccinimide esters, such as N-hydroxysuccinimide acetate.
  • acid chlorides such as acetic acid, propanoic acid, isobutyric acid, butanoic acid or benzoic acid chloride.
  • chloroformates mention may be made of 9-fluorenylmethyl chloroformate (Fmoc-Cl).
  • anhydrides mention may be made of acetic, propanoic, isobutyric, butanoic, benzoic, maleic, succinic or phthalic anhydride.
  • activated carbonate esters mention may be made of di-tert-butyl dicarbonate (Boc 2 0).
  • the masking reagent is acetic anhydride or its dry form, N-hydroxy-succinimide acetate.
  • an alkylating agent may be selected from the group consisting of alkyl halides, aryl sulfates, aryl diazo methyls, triazenes and aldehydes.
  • alkyl halides mention may be made of methyl iodide, ethyl or propyl, or methyl bromide.
  • aryl diazomethyl compounds mention may be made of methyl diazopyridine (WO2010012949A1).
  • aldehydes mention may be made of formaldehyde and acetaldehyde.
  • the acylating or alkylating reagent is used in combination with a catalyst known to those skilled in the art.
  • a catalyst known to those skilled in the art.
  • acylation with acetic anhydride for example 4-dimethylaminopyridine (DMAP) can be used as a catalyst.
  • DMAP 4-dimethylaminopyridine
  • the reagent used is chosen from amines, alcohols and thiols, for the masking of the carboxylic acid functions of the proteins and / or polysaccharides, in combination with a coupling agent.
  • the coupling agent is used to activate the carboxylic acid functions of the proteins and / or polysaccharides by forming activated esters. These activated esters will then react with a nucleophile selected from amines, alcohols and thiols.
  • the nucleophile may be present on the protein and / or the polysaccharide (amine, alcohol or thiol of an amino acid of the protein and / or polysaccharide) or in the medium, as in the case of a nucleophilic buffer, for example the Tris.
  • the coupling agent may for example be chosen from carbodiimides, among which mention may be made of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), di-isopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC). ).
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • DIC di-isopropylcarbodiimide
  • DCC dicyclohexylcarbodiimide
  • the carbodiimides may be used in combination with other reagents such as N-hydroxysuccinimide and triazoles, among which may be mentioned 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).
  • the masking reagent may also be bi-functionalized or multifunctionalized in order to establish bridges between the reactive residues of the proteins (bridging compounds such as bisaldehyde, N-hydroxysuccinimide bis ester, acid bis boron chloride and their derivatives). bridging compounds such as bisaldehyde, N-hydroxysuccinimide bis ester, acid bis boron chloride and their derivatives).
  • an acylating agent such as acetic anhydride may be used in concentrations of between 0.01 M and 1.8 M, for example between 0.1 M and 1.0 M, for example between 0.2 and
  • concentration of the masking reagent used for the treatment of the sample may be adjusted according to the concentration of proteins and / or polysaccharides in the sample, the masking reagent used, the source of the sample. sample and lysis conditions used.
  • concentration of acylating agent may be chosen to react with the amine and / or carboxylic acid functions of the proteins and / or polysaccharides, without reacting with the nucleic acids.
  • the incubation time of the lysate, or the sample being lysed when the lysis and treatment step are concomitant, in the presence of the masking reagent is generally between 1 second and 30 minutes, but preferably less than 10. minutes.
  • a step is performed to control the acidity of the treated lysate, for example with sodium hydroxide (NaOH), in particular in order to obtain a pH of the treated lysate of preferably between 5 and 7.
  • NaOH sodium hydroxide
  • the amount of sodium hydroxide added during this step is low, this step therefore only makes it possible to control the pH before the capture step, it is not a step of deprotection of the amine functions and / or masked carboxylic acids of the proteins and / or polysaccharides.
  • This step of controlling the acidity of the treated lysate is optional, in a particular embodiment, this step is not present.
  • a treated lysate comprising modified proteins and / or polysaccharides.
  • at least part of the amine and / or carboxylic acid functions of the proteins and / or polysaccharides are masked after treatment, preferably the nucleic acids are not modified by the masking reagent.
  • the capture step consists of placing the treated lysate in the presence of a solid support, for example a silica-based solid support, under conditions permitting the adsorption of the nucleic acids (solid phase extraction of the nucleic acids).
  • a solid support for example a silica-based solid support
  • the silica-based support for example silica beads or particles, makes it possible to separate the nucleic acids, retained by adsorption on the silica, from other cellular contaminants (membranes, proteins treated with the masking reagent, etc.). .
  • no antibody is used during the capture step.
  • the capture step takes place directly after the step of treating the sample or lysate with the masking reagent.
  • the sample comprises modified proteins and / or polysaccharides (i.e. masked by the masking reagent) and non-nucleic acids. modified (that is, not masked by the masking reagent).
  • a sufficient amount of silica beads or particles is added to the treated lysate.
  • the conditions for the adsorption of nucleic acids include the presence of chaotropic agents, such as guanidinium chloride or thiocyanate.
  • guanidinium in a buffered medium between pH 4 and pH 8, for example with the aid of organic compounds such as the salts of TRIS (Tri (hydroxyethyl) amino methane), the salts of acetate, of phosphate, of citrate, or of MES ("morpholino ethane sulfonic acid").
  • a detergent preferably the triton XI 00 or one of these analogs (Tergitol, Tween, Bridj, Nonidet, Ecosurf, etc.).
  • said detergent is contained in the lysis buffer.
  • the silica-based support is preferably negatively charged between pH 5.0 and pH 8.0.
  • chaotropic agents or alcohols are not used during the adsorption capture step on the solid support.
  • An appropriate support will then be used, in particular an amine support.
  • beads or particles of silicas that can be used for the capture step, mention may be made of the particles included in the following kits: NucliSENS easyMAG Magnetic Silica (BioMerieux), Film Array (BioMérieux / Biofire Diagnostic), kits Qiasymphony (Qiagen) and Magnapure Kit (Roche).
  • the silica-based solid support is an extraction column with a silica membrane.
  • a silica membrane By way of example, mention may be made of the kits sold under the names Purelink Genomic DNA Extraction Kit (Invitrogen), DNeasy Blood and Tissue Kit (Quiagen).
  • the treated lysate is deposited on the silica membrane column, centrifuged to pass the lysate through the column. Nucleic acids are retained on the column and proteins, polysaccharides and other cellular debris pass through the column.
  • silica-based carriers and their uses for the extraction of nucleic acids are described in particular in the following articles: Cady, et al. Nucleic acid purification using micro fabricated Silicon structures. Biosensors and Bioelectronics 19, 59-66 (2003); A. Melzak, CS Sherwood, RFB Tumer, CA Haynes. Driving Forces for DNA Adsorption to Silica in Perchlorate Solutions. J Colloid and Interface Science 181, 635-64 (1996); Tian, et al. Evaluation of Silica Resins for Direct and Efficient Extraction of DNA from Complex Biological Matrices in a Miniaturized Format, Analytical Biochemistry 283, 175-191 (2000); Wolfe, et al. Toward has a microchip-based solid-phase extraction method for isolation of nucleic acids. Electrophoresis 23, 727-733 (2002).
  • magnetic silica particles are used.
  • the magnetic silica particles may be held by a magnet during the washing and, where appropriate, elution steps following capture. This embodiment is particularly preferred for automation of the extraction process.
  • magnetic particles which may be used include the kit sold under the name NucliSENS easy M AG Magnetic Silica (BioMérieux).
  • the process for extracting nucleic acids from an unbound sample comprising proteins and / or polysaccharides comprises a step of capturing the nucleic acids by contacting the unfixed sample. with a suitable solid support, characterized in that, prior to the capture step, said method comprises a step of treating the sample with at least one reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides of the sample, and in that the capture step takes place directly after the step of treating the sample with the masking reagent.
  • washing steps can be carried out in the presence of a washing buffer, allowing the removal of contaminating elements without detaching the nucleic acids from the support.
  • the washing step may for example comprise a washing step in a suitable washing buffer, for example a weakly salt solution optionally containing an alcohol and / or a washing step in the presence of an alcohol in order to eliminate the salts.
  • at least one washing step is performed.
  • the elution step consists in releasing the nucleic acids retained on the solid support.
  • a buffer of basic pH for example a pH of between 8 and 10, and of low ionic strength, such as Tris or borate buffer, is used.
  • the detection or amplification step is carried out directly on the appropriate solid support having captured the purified nucleic acids without prior elution step. Preferably, this is done on a ball or a particle or a membrane and more preferably still on a magnetic particle or not.
  • nucleic acids thus purified by the method described above can be used in particular for amplification and in vitro detection applications.
  • the invention is particularly suitable for the preparation of nucleic acids for the detection of nucleic acids of interest in a sample (in vitro detection test), especially following the amplification of a nucleic acid of interest.
  • the invention therefore relates to a method for detecting a nucleic acid sequence of interest in a biological sample, said method comprising:
  • the detection step (ii) comprises a step of amplification of the nucleic acid sequence of interest.
  • the invention also provides a method of amplifying a nucleic acid comprising (i) carrying out a method for extracting nucleic acid on a suitable support as described above, and (ii) ) a nucleic acid amplification step using a DNA polymerase.
  • the amplification step can take place just after the capture step. Since the nucleic acids are not modified by the masking reagent, it is not necessary to unprotect them before proceeding with the amplification.
  • the amplification process is carried out using a DNA polymerase, such as Taq polymerase, Pfu polymerase or T7. polymerase, the Klenow fragment of E. coli DNA polymerase. coli and / or reverse transcriptase, or any other polymerase.
  • a DNA polymerase such as Taq polymerase, Pfu polymerase or T7. polymerase, the Klenow fragment of E. coli DNA polymerase. coli and / or reverse transcriptase, or any other polymerase.
  • the amplification method is a polymerase chain reaction (PCR) reaction, well known to those skilled in the art.
  • the PCR protocol comprises, for example, 20 to 40 cycles, each cycle comprising at least (i) a denaturation phase of the DNA to be amplified at a temperature generally of between 90 ° C. and 95 ° C., (ii) a phase of hybridization of the primers with the DNA to be amplified at a temperature generally between 55 ° C and 65 ° C and (iii) an extension phase at a temperature generally between 68 ° C and 75 ° C.
  • PCR nucleic amplification methods can also be implemented.
  • nested PCR nested PCR
  • quantitative or qPCR
  • semi-quantitative or real-time PCRs error-prone PCRs
  • reverse transcription PCR RT-PCR
  • LAMP LAMP
  • NASBA TMA
  • RP A LCR
  • RCR RCR
  • SR SR
  • RCA reverse transcription PCR
  • the invention also provides nucleic acid extraction kits or kits comprising at least i. a reagent for masking the amine and / or carboxylic acid functions of the proteins and / or polysaccharides as described in the preceding paragraphs, for example an acylating agent and preferably, acetic anhydride or its dry form, N-Hydroxy acetate succinimide
  • a solid support suitable for the extraction of nucleic acids iii. if appropriate, a catalyst of the reaction for masking the amine and / or carboxylic acid functions using the reagent, and / or
  • the extraction kit may further include buffers, controls and / or instructions for use.
  • the extraction kit does not include a deprotection reagent for amine functions and / or masked carboxylic acids.
  • the extraction kit does not include an organic solvent.
  • the kit may comprise in particular the following elements: i. a coupling agent, for example, EDC,
  • a suitable solid support based on silica for the extraction of nucleic acids, for example silica beads, and in particular magnetic silica beads, and at least one of the following optional elements:
  • a lysis buffer comprising Tris or another nucleophilic buffer, iv. one or more wash buffers, and / or,
  • the kit comprises a column with a silica membrane as a solid support based on silica.
  • Figure 1 Inhibition of the capture of DNA on solid support by certain proteins in chaotropic conditions
  • FIG. 2 Inhibition of the capture of the DNA on a solid support by certain polysaccharides under chaotropic conditions
  • Figure 3 Inhibition of the capture of DNA on solid support by model mono-functional polymers.
  • Figure 4 A. Extraction of the nucleic acids contained in a blood sample using the easyMAG extraction kit (BioMérieux) and pretreatment of the lysate with acetic anhydride (Ac20) or acetic acid (AcOH) (capture in 3M GuHCl + 1% Triton and ethanol washes). B. Measurement of the amount of nucleic acids by chromatographic analysis.
  • FIG. 5 HPLC analysis of extraction eluates of a mixture of DNA and RNA that is or is not subjected to acetic anhydride. Top: Eluate from the extraction of a mixture of 10 ⁇ g of DNA and RNA and 5.2 mg of protein in the presence of acetic anhydride. Bottom: Eluate obtained from the extraction of a mixture of 10 ⁇ g of DNA and RNA.
  • FIG. 6 Fluorescence detection curve (RFU) of the real-time amplification of CMV virus nucleic acids in the blood by PCR.
  • the curves of FIG. 6 correspond respectively to the control amplification carried out after extraction without a biological matrix (triangles), with a biological matrix without treatment with acetic anhydride (cross) and with a biological matrix with acetic anhydride treatment (round). .
  • Figure 7 Sensitivity of the detection of CMV virus in the blood, without or with treatment with Ac 2 anhydride prior to extraction (Ct): Left: 10 e 4 copies of CMV in Tris HCl, In the center : lO e 4 copies of CMV in the blood, right: lO e 4 copies of CMV in the blood + AC20.
  • Figure 8 Extraction of DNA in the presence of a mixture of hemoglobin and human serum albumin on a column with silica membrane with and without treatment with Ac20. Left (dark gray): without Ac20 treatment, right (light gray): with Ac20 treatment
  • the LC-MS analyzes were carried out with a WATERS Alliance 2795 HPLC chain equipped with a PDA 996 diode array detector (Waters ), a ZQ 2000 mass spectrometry detector (Waters), an Empower version 2 software.
  • the ZQ 2000 mass spectrometer has an Electrospray ionization source. Ionizations were performed in positive mode with a cone voltage of 20V and a voltage at the level of the capillary of 3.5kV.
  • Example la Demonstration of the Inhibitory Effect of Protein Compounds of the Blood for the Capture of Nucleic Acids on a Solid Support (Magnetic Silica Particles Used at pH 7)
  • the inventors have assumed that the first step was to attempt to increase the performance of the capture step.
  • the inhibitory effect seems to be partly related to the isoelectric point of the proteins, with, respectively, in order of inhibition, the human serum albumin (HSA) which has an isoelectric point of 4.8, the fibrinogen of 5.8, the hemoglobin and immunoglobulin 7, and lysozyme of 11.3.
  • HSA human serum albumin
  • This example thus demonstrates the inhibitory effect of the compounds of the blood on the extraction yield of the nucleic acids, and more particularly the compounds proteins that are negatively and positively charged. It is found that the compounds among the most competitive are those present in large quantities in the blood (human serum albumin, fibrinogen, immunoglobulins and hemoglobin).
  • a blood sample could contain between 200 and 500 mg / ml of proteins.
  • the sample could comprise at least 40 to 100 mg of "inhibitor" nucleic acid capture compounds.
  • Example 3 Inhibitory effect of high protein concentration for nucleic acid extraction, DNA elution efficiency and purity of the extracted DNA (magnetic silica particles used at pH 7)
  • the lysis solution corresponds respectively to 23 ⁇ g of DNA in 200 ⁇ l of GuHCl or 3 M GuSCN, 50 mM Tris pH 7, 1% Triton XI 00, or easyMAG lysis buffer (bioMérieux, Marcy l'étoile, France). diluted to 3M.
  • Table 1 Extraction yield of 23 ⁇ g of DNA in the presence or absence of protein inhibitors. Figures in parentheses indicate the 260/280 nm ratio of the eluate. As in the previous examples, a sharp drop in the extraction yield is observed. Whatever the lysis conditions, we go from a DNA extraction yield (capture, washing, elution) of about 60%, to less than 6% when we add proteins in the medium.
  • the purity of the extracted nucleic acids is also greatly affected since the presence of proteins lowers the 260/280 representative ratio of protein contamination in the final eluate from 2 to less than 1.6 on average (see Table 1). This therefore reflects a very high protein concentration in the eluate.
  • EXAMPLE 4 Increase of the extraction yield of the DNA by the use of a reagent for masking the amine and / or carboxylic acid functions in the presence of a high concentration of proteins (use of magnetic particles at pH 7)
  • the inventors have performed the same experiment as that described in Example 3 above but this time adding a treatment with acetic anhydride or EDC prior to contact with the magnetic silica particles.
  • the total extraction yield is calculated relative to the initial 23 ⁇ g of DNA and the amount of nucleic acids obtained in the eluate.
  • the assay is performed by UV spectrophotometry at 260 nm.
  • GuHSCN which has a higher denaturing power than GuHCl, allows a better yield of DNA capture in the presence of proteins.
  • the presence of EDTA in the LB decreases the 260/280 ratio, while the Triton X-100 also present in the LB has a very beneficial effect.
  • Table 2 Extraction yield of 23 ⁇ g of DNA in the presence or absence of protein inhibitors and with or without acetic anhydride (Ac20) or EDC, and under different lysis conditions. Numbers in parentheses indicate the 260/280 nm ratio of the eluate.
  • Example 5 Improvement of the efficiency of the extraction of nucleic acids in the blood (extraction with magnetic particles at pH 7)
  • the blood sample was subjected during the lysis step to treatment with 0.3 M acetic anhydride or 0.3M acetic acid for 5 min, before being neutralized by adding a few m ⁇ of sodium hydroxide. (in comparison with an untreated reference sample).
  • the magnetic particles were added and the lysate put on the easyMAG following the extraction protocol of the supplier (bioMérieux).
  • the amount of nucleic acids present in the blood is measured by UV spectrophotometry (Nanodrop, Thermofischer). It is noted in a remarkable way in FIG.
  • the hydrolysates were analyzed by HPLC so as not to be impeded by the proteins and the area corresponding to the nucleosides (DNA and RNA) was measured on the chromatograms by integration of the masses corresponding to the nucleosides.
  • the results obtained show that the sample of blood previously treated with acetic anhydride contains 12 times more nucleic acids than the untreated sample or treated with acetic acid (which is non-acylating) (FIG. 4B).
  • a l33pL a blood solution are added 237 pL lysis buffer (6M GuHCl, 15% Triton, NaOAc, pH 5.5) and 6PL, 12m1 or 24 pL of Ac 2 0.
  • the medium is incubated with stirring for 3 to 5 min at the thermomixer at 25 ° C and 500 rpm before rectifying the pH to 5.5 if necessary by adding a few m ⁇ of 10M NaOH.
  • 3 ⁇ L, 4.5 and 6.8 ⁇ L of NaOH are added for 6 ⁇ L, 12 ⁇ L and 24 ⁇ L of Ac 2 O, respectively.
  • the lysate is added to the magnetic silica particles in order to extract the nucleic acids.
  • wash solutions (3 times 70 ml Wash Buffer IX) precede the elution step which takes place in a solution of 70 ⁇ l of Tris HCl with stirring at 1400 rpm, at 70 ° C. in 5 minutes.
  • the lysates are subjected to nucleic acid hydrolysis enzymes as described in Example 5 to determine the amount of nucleic acid extracted.
  • the results obtained show that the addition of the masking reagent (acetic anhydride) in the presence of blood makes it possible to double the quantity of nucleic acids extracted.
  • Example 7 Treatment of DNA with acetic anhydride under the conditions of the process according to the present disclosure does not alter the chemical structure of the nucleosides
  • Example 8 Demonstration that the structure of DNA and RNA is not affected during treatment with acetic anhydride under nucleic acid extraction conditions
  • Example 7 The same demonstration as in Example 7 was carried out using this time a mixture of DNA and RNA which was brought into the presence or absence of acetic anhydride and then extracted with magnetic silica particles. Enzymatic hydrolysis of these eluates has unambiguously confirmed that there are no chemical modifications of the nucleosides following the action of acetic anhydride (see Figure 5). Thus, it can be concluded that the nucleic acids thus extracted should be amplifiable by PCR or by any other enzymatic amplification reaction of genetic material.
  • Example 9 Acetic anhydride treatment of a blood sample makes it possible to detect the CMV virus more sensitively
  • a 200 ml sample of blood containing CMV virus (Cytomegalovirus in culture) with 10 copies is added to 800 ml of lysis buffer (6M GuHCl, 15% Triton, AcONa, pH 5.5) to which 9 ml of pure acetic anhydride and, optionally Im ⁇ 10M NaOH, after a few minutes of reaction.
  • a control is carried out with blood where there is no prior treatment with acetic anhydride, and another without blood sample, corresponding to 63 ml of Tris HCL pH7 containing 104 copies of CMV and then mixed with 237 ml of buffer. lysis.
  • the nucleic acids contained in the lysate are then extracted and purified as described in Example 6.
  • the eluate is then subjected to real-time PCR amplification using a RGene CMV kit (kit CMV 69-003B: lot 1005408010, BioMérieux, Marcy L'Etoile, France) and a thermocycler (CFX96 TouchTM BioRad) to determine the detection sensitivity of extracted nucleic acids expressed in number of PCR cycle (Ct).
  • This number is a function of the concentration of nucleic acids, the lower it is and the easier it is to detect the nucleic acids of the pathogen because the initial concentration of nucleic acids is high.
  • There is a scaling factor of 10 between each Ct logarithmic scale).
  • the curves of FIG. 6 correspond respectively to the control amplification carried out after extraction without a biological matrix (triangles), with a biological matrix without treatment with acetic anhydride (cross) and with a biological matrix with acetic anhydride treatment (round). .
  • the number of PCR cycles is therefore at 34 Ct when the CMV virus has been extracted without a biological matrix, then goes to 38 Ct when the same amount of nucleic acids is extracted from a sample containing blood. This shows that the inhibitors contained in the biological matrix are responsible for the loss of more than 99.9% of the initial nucleic acids.
  • the number of Ct returns to 34 is equivalent to a sample without matrix when the sample previously treated with acetic anhydride.
  • Figure 7 is another representation of Figure 6 with the accuracy on Ct.
  • the inventors have used a nucleic acid extraction medium different from magnetic silica particles, in order to demonstrate that acetic anhydride is also of interest in other extraction technologies such as extraction columns.
  • Silica membrane base Silica membrane base.
  • 10 ⁇ g of salmon sperm DNA (20 kB) are mixed with 300 ml of a solution of 3M GuHCl, 50 mM AcO-Na pH 5.5 and 30% ethanol with increasing concentrations of a mixture of hemoglobin and human serum albumin at a level of 0.300, 600, 1200, 1800 and 2400 ⁇ g (with a hemoglobin / HSA ratio of 3/1).
  • This mixture is treated or not with 6m1 of acetic anhydride for a few minutes (10) with thermomixer stirring.
  • the mixture is neutralized with 1 ml of 5M NaOH to restore the pH to 5.5.
  • the mixture is deposited on a QiaQuick column (Qiagen, Heiden, Germany) and centrifuged for 6 minutes at 13,200 rpm.
  • the column is washed 3 times with 200 ⁇ l of MES 50 mM pH 5.5 / 80% ethanol by centrifuging at 13,200 rpm for 3 minutes.
  • the immobilized nucleic acids are then eluted with 200 ml of borate buffer pH 8.5 at 3 mM for the first time, then this solution is ironed in order to elute all the nucleic acids.
  • the eluate is subjected to enzymatic hydrolysis and the extracted nucleic acids are quantified by chromatographic analysis.
  • Figure 8 shows a significant improvement in the amount of DNA extracted when the sample was subjected to acetic anhydride.
  • Example 11 Demonstration that the use of acetic anhydride according to the invention also allows the inhibition of nucleases
  • a model oligonucleotide of 60 bases (1 nmol) is brought into contact with 0.01 unit of nuclease P1 in 48 ml of water and 2 ml of 500 mM MES pH 4.7 by adding or not adding 6 ml of pure acetic anhydride.
  • the results are shown in FIG. 9.
  • chromatogram A Compared to the control oligonucleotide which has undergone no treatment (chromatogram A), in the case where there is no acetic anhydride, the oligonucleotide is immediately hydrolyzed by nuclease (chromatogram B).
  • chromatogram C On the other hand, in the case where acetic anhydride has been added, there is very little degradation of the oligonucleotide (chromatogram C). This therefore demonstrates that acetic anhydride allows the inactivation of nuclease by alkylating it.
  • Example 12 Demonstration that the reagent can react selectively on proteins under chaotropic conditions (case of acetic anhydride)
  • Figures 10 and 11 show the monitoring by HPLC (Conditions B) of the acetylation of HSA or hemoglobin at 25 mg / ml with acetic anhydride between 0 and 1.8 M under lysis conditions (GuHCl 3M or GuSCN 3M, 50 mM Tris HCl pH 7).
  • acetic anhydride between 0 and 1.8 M under lysis conditions
  • the concentration of acetic anhydride that the solid corresponding to the protein widens and moves to the right, meaning that the protein becomes more hydrophobic which is the result of its acylation.
  • the retention time no longer changes, which means that all the nucleophilic groups of the protein have reacted.
  • the hemoglobin acetylation monitoring chromatograms that the heme is not modified by acetylation and does not undergo any change in its retention time.
  • acetic anhydride can react rapidly and efficiently on proteins under lysis conditions and in the presence of nucleophilic compounds such as Tris.
  • acetic anhydride can acetylate a protein in an aqueous medium while being selective is really a surprise since the majority of the reactions with acetic anhydride are described in an organic solvent. Due to the presence of water, tris and guanidine which are susceptible to the hydrolyzer, it was not obvious that the acetic anhydride can react at low concentration with a protein.

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