阅读说明：本技术 提取核酸的方法 (Method for extracting nucleic acid ) 是由 阿兰·劳伦 阿诺·布尔 阿里·拉尤恩 于 2019-03-15 设计创作，主要内容包括：本发明涉及使用例如基于二氧化硅的合适的固体载体提取核酸的方法。特别地,所述方法包括通过使样品与合适的固体载体接触来捕获核酸的步骤,其特征在于所述方法包括在捕获步骤之前用至少一种试剂处理样品以掩蔽样品的蛋白质和/或多糖的胺或羧酸官能团的步骤。(The present invention relates to a method for extracting nucleic acids using a suitable solid support, for example based on silica. In particular, the method comprises a step of capturing the nucleic acids by contacting the sample with a suitable solid support, characterized in that it comprises a step of treating the sample with at least one reagent to mask the amine or carboxylic acid functions of the proteins and/or polysaccharides of the sample, prior to the capturing step.)

1. A method for extracting nucleic acids from a sample comprising proteins and/or polysaccharides, said method comprising a step of capturing the nucleic acids by contacting the sample with a suitable solid support, for example a silica-based solid support, characterized in that it comprises a step of treating the sample with at least one reagent to mask the amine and/or carboxylic acid functional groups of the proteins and/or polysaccharides of the sample before the capturing step.

2. The process according to claim 1, wherein the agent for masking amine functions is selected from acylating or alkylating agents.

3. The process according to claim 1 or 2, wherein the masking agent is selected from acylating agents of formula (I):

wherein R is an organic group, an organoxy group or an organoamino group,

LG is a leaving group selected from halogen, organoxy and organoamino.

4. The method according to any one of claims 1 to 3, wherein the masking agent is an acylating agent selected from the group consisting of activated esters, acyl halides, chloroformates, anhydrides, activated carbonates and carbonyldiimidazole.

5. The method according to any one of claims 1 to 4, wherein the masking agent is an anhydride selected from acetic anhydride, propionic anhydride, isobutyric anhydride, butyric anhydride or benzoic anhydride.

6. The method of claim 1 or 2, wherein the masking agent is an alkylating agent selected from the group consisting of alkyl halides, diazo compounds, and aldehydes.

7. The method according to claim 1 or 2, wherein the masking agent is selected from the group consisting of amines, alcohols and thiols, used in combination with a coupling agent for masking the carboxylic acid functions of the protein.

8. The method of claim 7, wherein the coupling agent may be selected from carbodiimides, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), Diisopropylcarbodiimide (DIC), and Dicyclohexylcarbodiimide (DCC).

9. The method according to any one of the preceding claims, for extracting nucleic acids from a biological sample, and comprising the steps of:

a. lysing the cells, for example by contacting the biological sample with a lysis buffer,

b. Treating the lysate with a reagent that masks the amine and/or carboxylic acid functional groups of the proteins and/or polysaccharides of the sample,

c. capturing the nucleic acid by contacting the treated lysate with a suitable solid support, e.g.a silica-based solid support,

d. washing the support with a washing buffer, where appropriate, and

e. where appropriate, the nucleic acid is eluted.

10. The method according to any one of the preceding claims, wherein a suitable solid support consists of silica particles, in particular magnetic silica particles.

11. The method according to any one of claims 9 or 10, wherein the biological sample is a sample of blood, plasma or serum.

12. The method of any one of claims 9 to 11, wherein the capturing step is performed in the presence of a chaotropic agent.

13. The method of any one of claims 9 to 12, wherein the method does not comprise removing protein using a protease.

14. The method of any one of claims 9 to 13, wherein no organic solvent is added prior to or during the nucleic acid capture step.

15. The method according to any one of claims 9 to 14, wherein the method comprises an additional step of detecting a target nucleic acid, in particular by amplifying the extracted nucleic acid.

16. A nucleic acid extraction kit at least comprises

i. An agent for masking amine and/or carboxylic acid functional groups of proteins and/or polysaccharides as defined in any of claims 1 to 8,

a solid support suitable for nucleic acid extraction, such as a silica-based solid support,

a catalyst for the reaction of masking the amine and/or carboxylic acid functional groups with a reagent, where appropriate,

where appropriate, a coupling agent.

17. The nucleic acid extraction kit of claim 16, wherein the masking agent is acetic anhydride or a dried form thereof, N-hydroxysuccinimide acetate.

18. Use of an agent for masking amine and/or carboxylic acid functional groups of a protein and/or polysaccharide as defined in any one of claims 1 to 8 for inhibiting nucleases.

19. Use according to claim 18, wherein the nuclease is comprised in a lysate obtained by lysing a biological sample, e.g. for amplifying and/or detecting nucleic acids in a sample.

Prior Art

The rise in molecular biology has led to great advances in diagnostics. From the test sample, nucleic acids belonging to the host or infectious microorganism contained in the sample can be extracted and detected. The detection or even quantification of such genetic material makes it possible to establish a diagnosis concerning microbial infection or the presence of oncogenes. This is typically accomplished in three steps as follows:

1) Nucleic acids are extracted from complex biological samples (blood, tumors, food, etc.) which involve chemical or mechanical lysis of the cells to release the cell contents, in particular the nucleic acids. If their amount is not sufficient for direct detection, they are selectively purified and subsequently amplified.

2) The purified nucleic acid is amplified by the DNA amplification technique NASBA, RT-PCR, PCR or the like. This step is necessary when the amount of nucleic acid collected from the biological sample is very low or the sensitivity of the test is insufficient for direct detection.

3) The amplified nucleic acids are detected by a technique known as endpoint, real-time, sequencing, and the like. This step enables selective quantification of the target nucleic acid tested, depending on the detection technique used.

For the sensitive and specific detection of nucleic acids, in particular for the diagnosis to be carried out as accurately as possible, it is of great importance to extract and/or isolate nucleic acids (DNA and RNA) from cells in an efficient manner. This extraction and/or purification step is often critical, as this first step will determine the quality of the series of events that will yield the final result of the diagnostic test. Therefore, it is necessary to have a nucleic acid extraction method that is as specific and efficient (in terms of amount, purity and time) as possible, so as not to lose information that could lead to misdiagnosis and be fatal to the patient.

Many techniques have been developed to attempt to extract nucleic acids from different biological samples. The initial process involves a number of steps, typically including enrichment of cells containing nucleic acids, lysis of these cells, separation and removal of proteins, membranes and other cellular components, and purification of the remaining nucleic acids by precipitation in organic solvents. These techniques are expensive, time consuming and often cannot be automated. Therefore, they are no longer suitable for the current practice that requires automation to obtain results quickly and to avoid contamination and human error, especially in the case of sepsis "blood infection" that threatens patient survival.

Recent nucleic acid extraction techniques use a solid phase in which cells are lysed under specific reaction conditions and the released nucleic acids are bound to the solid phase. As is well known in the art, current nucleic acid extraction techniques often employ a solid phase, such as silica-coated particles. Silica has the property of reversibly adsorbing nucleic acids at a certain salt concentration and pH, which makes 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, page 495, or in U.S. Pat. No. 5234809 of the same authors.

It is also known to use silica coated magnetic particles. The magnetic portion of the particles is most often used to facilitate and automate the capture, washing and elution steps of nucleic acids, as a simple magnet can displace the particles in the tube and collect the supernatant to perform the washing step. The extraction yield of nucleic acid is obviously improved. These techniques are described in "Magnetic moieties for the separation and Purification of Nucleic Acids", S.Berensmeier, Applied MicroBiotechnology 200673495-; "The use of magnetic nanoparticles in The definition of new molecular detection systems", I.J.Bruce, Journal of nanosciences and Nanotechnology (2006), page 6(8) 2302-2311; and "Optimization of fluidic factors of nucleic acid adsorption on to silicon-coated magnetic particles" -Application to viral nucleic acid extraction from server ", Ning Sun et al, Journal of Chromatography A,2014,1325, 31-39.

The present disclosure is directed to methods for extracting nucleic acids that meet one or more of the following criteria:

1) the implementation is simple, and the operation is simple,

2) can be carried out on biological samples of different origins,

3) the process is carried out in a substantially aqueous medium,

4) The extraction yield is good, and the extraction yield is good,

5) the purity level of the extracted nucleic acid is high,

6) the possibility of automation of the process of the present invention,

7) the quality of the DNA is sufficient for the step of amplifying and/or detecting the target sequence, in particular in the case of in vitro diagnostic tests, and/or

8) Better detection sensitivity and more reliable results.

Disclosure of Invention

The present disclosure relates to a method for extracting nucleic acids from a sample comprising proteins and/or polysaccharides, said method comprising a step of capturing the nucleic acids by contacting the sample with a suitable solid support, characterized in that said method comprises a step of treating the sample with at least one reagent to mask the amine and/or carboxylic acid functional groups of the proteins and/or polysaccharides of the sample before the capturing step. In a particular embodiment, the agent for masking amine functions is selected from acylating or alkylating agents. For example, the masking agent is selected from acylating agents of the formula (I):

wherein R is an organic group, an organoxy group or an organoamino group,

LG is a leaving group selected from halogen, organoxy and organoamino.

More specifically, the masking agent may be an acylating agent selected from the group consisting of activated esters, acyl halides, chloroformates, anhydrides, activated carbonates, and carbonyldiimidazole. In another embodiment, the masking agent is an anhydride selected from acetic anhydride, propionic anhydride, isobutyric anhydride, butyric anhydride or benzoic anhydride.

In another embodiment, the masking agent is an alkylating agent selected from the group consisting of alkyl halides, diazo compounds, and aldehydes.

In another embodiment, the masking agent is selected from the group consisting of amines, alcohols, and thiols, used in combination with a coupling agent to mask the carboxylic acid functionality of the protein. Carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), Diisopropylcarbodiimide (DIC) or Dicyclohexylcarbodiimide (DCC) may be mentioned as examples of coupling agents.

In a particular embodiment which may be combined with the above-mentioned embodiments, the method for extracting nucleic acids from a biological sample comprises the steps of:

a. lysing the cells, for example by contacting the biological sample with a lysis buffer,

b. treating the lysate with a reagent that masks the amine and/or carboxylic acid functional groups of the proteins and/or polysaccharides of the sample,

c. capturing the nucleic acid by contacting the treated lysate with a suitable solid support, such as a silica-based support,

d. washing the support with a washing buffer, where appropriate, and

e. where appropriate, the nucleic acid is eluted.

Generally, in the process defined above, the solid support may consist of silica particles, in particular magnetic silica particles.

In a particular embodiment which may be combined with the above embodiments, the biological sample is a sample of blood, plasma or serum.

In a particular embodiment, which may be combined with the above embodiments, the capturing step is performed in the presence of a dispersing agent.

In a particular embodiment, which can be combined with the above embodiments, the extraction method does not comprise the use of a protease to remove the protein.

In a particular embodiment, which can be combined with the above embodiments, no organic solvent is added before or during the nucleic acid capture step. Advantageously, less than 50. mu.L, preferably less than 10. mu.L, even more preferably less than 1. mu.L of organic solvent is added per 100. mu.L of sample before or during the nucleic acid capture step.

In a particular embodiment which can be combined with the above embodiments, the method comprises an additional step of detecting the target nucleic acid, in particular by amplifying the extracted nucleic acid.

The present disclosure also relates to a nucleic acid extraction kit comprising at least:

i. reagents for masking amine and/or carboxylic acid functional groups of proteins and/or polysaccharides as defined above, ii. a suitable solid support for extracting nucleic acids, for example a silica-based solid support,

A catalyst for the reaction of masking the amine and/or carboxylic acid functional groups with a reagent, where appropriate,

where appropriate, a coupling agent.

The present disclosure also relates to the use of an agent for masking amine and/or carboxylic acid functional groups of a protein and/or polysaccharide as defined above for nuclease inhibition. For example, the nuclease to be inhibited is contained in a lysate obtained by lysing a biological sample, e.g., for amplifying or detecting nucleic acids in the sample.

Definition of

"extraction" is understood to mean a technique for isolating nucleic acids from any sample, for example the isolation of DNA and/or RNA from eukaryotic cells, prokaryotic cells, human animal cells, microbial cells or tissue cells. Thus, in the sense of the present invention, extraction from a biological sample generally comprises cell lysis and purification of nucleic acids from the lysate.

The purification itself comprises the capture of the nucleic acids on a suitable solid support, preferably a silica-based solid support, and washing, possibly followed by elution of the nucleic acids. Trapping comprises adsorbing the nucleic acid on a solid support, and in the case where elution occurs, desorbing or releasing the nucleic acid from the solid support.

"sample" is understood to mean any type of sample comprising at least nucleic acids and an agent inhibiting the extraction of nucleic acids, such as proteins and/or polysaccharides. The sample may be of various origins, such as a dietary origin, an environmental origin, a human origin, a veterinary origin, or a cosmetic origin. All these samples, if not liquid, were pre-treated to liquid form. Thus, the sample used in the extraction process is in liquid form. Preferably, the sample used in the extraction process is an unfixed sample. "unfixed sample" is understood to mean a sample which has been stored in its original state without treatment. The fixation of the sample is a technique well known to the person skilled in the art and can be carried out, for example, with aldehydes such as formaldehyde or glutaraldehyde.

Examples of samples of dietary sources include, but are not limited to, samples of milk products (yogurt, cheese, etc.), meat, fish, eggs, fruits, vegetables, beverages (milk, fruit juices, sodas, etc.). Of course, samples of these dietary sources may also be from sauces or more refined dishes, or from raw or partially processed raw materials. Dietary samples may also be derived from animal feed, such as feed cake, meat and bone meal.

As described above, the sample may be an environmental source sample, and may include samples such as surface samples, water samples, and the like.

The sample may also comprise a biological sample of human or animal origin, which may correspond to a sample from a biological fluid (urine, whole blood or derivatives, such as serum or plasma, sputum or saliva, pus, cerebrospinal fluid, etc.), stool (e.g. cholera-like diarrhea), a sample from a nose, throat, skin, wound, organ, isolated tissue or cells, swab specimen, bronchoalveolar lavage or specimen, biopsy specimen. This list is obviously not exhaustive.

The term "sample" generally refers to a portion or quantity, more particularly a small portion or quantity, collected from one or more entities for analysis. The sample may have been subjected to pre-treatment including steps such as mixing, dilution or crushing, particularly if the initial entity is solid.

Typically, the sample after analysis may contain-or is suspected of containing-at least some nucleic acid representative of the presence of a microorganism, representative of a condition in the patient (e.g. immunosuppression, pregnancy, etc.) or representative of a disease to be detected, characterized or monitored.

In a particular embodiment, the sample comprises proteins having an isoelectric point of less than 8 or even less than 7. In particular, in a particular specific embodiment, the sample comprises at least one of the following proteins: human Serum Albumin (HSA), fibrinogen, immunoglobulins, in particular IgG and haemoglobin.

"microorganism" is understood to mean some or all of a bacterium, fungus, yeast, protozoa or virus.

"nucleic acid" is understood to mean all types of DNA or RNA: 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, in single-or double-stranded form.

"protein" is understood to mean any molecule comprising at least one polypeptide chain, characterized by a sequence of amino acid residues joined together by peptide bonds. This includes in particular peptides and polypeptides resulting from post-translational or other modifications as well as any modified polypeptides or derivatives thereof, their degradation products, in particular products degraded enzymatically, lipoproteins etc.

"suitable solid support" is understood to mean any support capable of participating in the extraction of nucleic acids from a biological sample. In a preferred embodiment, it is a carrier comprising or consisting of one of magnetic or non-magnetic silica or derivatives thereof (silicates, glass, silica modified with organic groups, etc.) which is capable of participating in the extraction of nucleic acids from a biological sample. It may also be a carrier based on paper, cellulose or pure magnetite or other polymers known for nucleic acid extraction. It can be at least a planar carrier, a hollow carrier, a wafer, a needle, a membrane, a plate, a sheet, a cone, a tube, a fiber, a bead, a particle, etc. The solid support is preferably a bead or particle or a membrane. The solid support is preferably magnetic.

In one embodiment of the method for extracting nucleic acids from a biological sample, the method comprises the steps of:

a) lysing the cells, e.g., by contacting the biological sample with a lysis buffer,

b) treating the lysate with an agent that masks the amine and/or carboxylic acid functional groups of the protein and/or polysaccharide of the lysate,

c) capturing the nucleic acid by contacting the treated lysate with a suitable solid support,

d) where appropriate, the support is washed with a wash buffer and the nucleic acids are eluted.

Advantageously, step b) is followed by step c), in other words there is no intermediate step between steps b) and c), for example no step of deprotecting the masked functional groups. In a particular embodiment, the method for extracting nucleic acids does not comprise a step of deprotecting the masked amine and/or carboxylic acid functional groups.

The present disclosure also relates to the use of an agent for masking amine and/or carboxylic acid functional groups of proteins and/or polysaccharides in a method for extracting nucleic acids from a biological sample using a suitable solid support.

The steps of the method are described in more detail below.

Cracking step

The lysis step involves rupturing the sample cells (cell wall and membrane) to release the nucleic acids. There are several methods of lysis: mention may in particular be made of mechanical lysis (for example with beads and/or ground material), chemical lysis or enzymatic lysis or lysis by heat shock.

In a particular embodiment, the cells are chemically lysed by contacting the sample with a lysis buffer. The lysis buffer must effectively rupture the cell membrane and be sufficiently gentle to avoid degradation of the nucleic acids.

The lysis buffer may comprise, for example, a detergent, and where appropriate, a chelating agent. The pH is typically maintained at 4 to 8, for example 6 to 8, using a suitable buffer, for example Tris HCl, optionally at a concentration of 10mM to 100 mM.

The detergent may be selected from tween, Triton, SDS and other detergents typically used at concentrations of 0.05% to 20%.

Optionally, the lysis buffer comprises reagents for inactivating the nuclease and/or for removing proteins, e.g., proteases, such as proteinase K. Other enzymes, such as lyases (hydrolases, zymozymes, etc.) may also be used to digest the walls of yeasts and fungi.

In a specific embodiment, the lysis buffer does not comprise reagents for removing proteins, in particular proteases. Indeed, the inventors have shown that the reagents used in the extraction method according to the present disclosure for masking amine and/or carboxyl functions are also advantageously able to inhibit the enzymatic activity of nucleases that may have been released into the lysate.

In a specific embodiment, the lysis buffer further comprises a chaotropic agent. The discretizing agents interfere with weak (non-covalent) intramolecular interactions such as hydrogen bonding, van der waals forces, and hydrophobic forces. Among the dispersing agents, mention may be made of urea, guanidine salts, such as guanidine hydrochloride or guanidine thiocyanate, and lithium perchlorate. They are usually used in concentrations of 1M to 6M, especially for GuSCN and GuHCl.

In another embodiment, the lysis buffer does not comprise a chaotropic agent.

It is also possible to add chelating agents, such as EDTA or similar compounds in a concentration of 5mM to 50mM, and/or reducing compounds, such as DTT (dithiothreitol) or TCEP (tricarboxyethylphosphine) or β -mercaptoethanol in a concentration of 0.5mM to 100 mM.

The lysis buffer may also comprise an organic solvent, such as an alcohol (ethanol, isopropanol, etc.). However, in a preferred embodiment, the lysis buffer does not comprise an organic solvent.

The chemical properties of the sample must be taken into account when optimizing the lysis buffer. For example, the acidity of a sample (e.g., some soil samples) may damage nucleic acids, and thus may be neutralized to ensure proper production of nucleic acids. The sample is contacted with the lysis buffer for a sufficient time, e.g., 0 to 15 minutes, to lyse the cells without damaging the nucleic acids.

Treatment of samples or lysates with masking agents

An essential feature of the method for extracting nucleic acids according to the present disclosure relates to the step of treating the lysate with an amine and/or carboxylic acid functional reagent that masks the proteins and/or polysaccharides of the sample. This treatment step may be performed after the lysis step by adding a masking agent to the lysate. Alternatively, the masking agent is directly contained in the lysis buffer and the lysis and treatment of the sample are performed simultaneously. The aim is to neutralize the charged functional groups of the proteins and/or polysaccharides, which may affect the extraction yield by solid supports, preferably silica-based solid supports. Indeed, in the following examples, the inventors have demonstrated the effect of biomolecules on the extraction yield, in particular on the proteins and/or polysaccharides of a biological sample, in an extraction process using solid supports, such as silica-coated particles. The proteins and/or polysaccharides may interact with the silica beads and/or nucleic acids, in particular via their charged functional groups, in particular their amine and/or carboxylic acid functional groups.

The inventors then conceived of the use of agents to mask these charged functional groups, in particular the amine and/or carboxylic acid functional groups of proteins and/or polysaccharides, to block this inhibitory effect of biomolecules. Thus, a "masking agent" is understood to mean any compound capable of reacting (preferably irreversibly reacting) with at least some of the amine and/or carboxylic acid functions of proteins and/or polysaccharides present in the lysate of a biological sample, so as to modify the polarity, isoelectric point and/or charge properties of these proteins and/or polysaccharides. Treatment with a masking agent makes it possible to mask the charged functional groups, in particular the amine and/or carboxylic acid functional groups of the proteins and/or polysaccharides. However, in a specific embodiment, the nucleic acid is not modified by the masking agent. The reaction conditions, in particular the concentration of the masking agent, may be chosen such that the nucleic acid is not modified in the masking step. In a particular embodiment, when a masking or coupling agent is present, it is present at a concentration of 0.01M to 1.8M, such as 0.1M to 1.0M, for example 0.2M to 0.6M.

In a particular embodiment, the masked amine and/or carboxylic acid functional groups of the modified protein and/or polysaccharide remain masked throughout the process for extracting nucleic acids according to the present disclosure. In this case, the method for extracting nucleic acids does not comprise a step of deprotecting the masked amine and/or carboxylic acid functions.

In a particular embodiment, the masking agent is selected from acylating or alkylating agents, which are capable of masking the amine function of the protein or polysaccharide. Preferably, the acylating or alkylating agent must be capable of masking the amine functionality of the protein or polysaccharide in the aqueous medium.

Generally, acylating agents that can be used as masking agents in the process for extracting nucleic acids have the following formula (I):

wherein the content of the first and second substances,

r is an organic group, an organoxy group or an organoamino group;

LG is a leaving group.

"leaving group" is understood to mean an atom or group which is removed from the carbon atom to which it is bound during the acylation reaction.

In one embodiment, LG is a leaving group selected from halogen, organoxy and organoamino.

In a particular embodiment, the masking agent may be an acylating agent selected from the group consisting of activated esters, acyl halides, chloroformates, anhydrides, activated carbonates and carbonyldiimidazole. Among the activated esters, mention may be made of tetrafluorophenylacetic or pentafluorophenylacetic acid esters, nitrophenylacetic acid esters, pentafluorophenyltrifluoroacetic acid esters and N-hydroxysuccinimide esters, such as N-hydroxysuccinimide acetate. Among the acyl halides, mention may be made of acyl chlorides, such as acetyl chloride, propionyl chloride, isobutyryl chloride, butyryl chloride or benzoyl chloride. Among the chloroformates, 9-fluorenylmethyl chloroformate (Fmoc-Cl) may be mentioned. Among the anhydrides, mention may be made of acetic anhydride, propionic anhydride, isobutyric anhydride, butyric anhydride, benzoic anhydride, maleic anhydride, succinic anhydride or phthalic anhydride. Among the activated carbonates, mention may be made of di-tert-butyl dicarbonate (Boc) ₂O)。

In a particularly preferred embodiment, the masking agent is acetic anhydride or a dry form thereof, N-hydroxysuccinimide acetate.

In another specific embodiment, the alkylating agent may be selected from alkyl halides, aryl sulfates, aryldiazomethyl groups, triazenes, and aldehydes. Among the alkyl halides, methyl iodide, ethyl or propyl iodide, or methyl bromide may be mentioned. Among the aryldiazomethyl compounds, mention may be made of methyldiazopyridine (WO2010012949A 1). Among the aldehydes, mention may be made of formaldehyde and acetaldehyde.

In one embodiment, the acylating or alkylating agent is used in combination with a catalyst known to those skilled in the art. In the case of acylation with acetic anhydride, for example, 4-Dimethylaminopyridine (DMAP) can be used as a catalyst.

In another embodiment, the reagent used is selected from the group consisting of amines, alcohols and thiols, in combination with a coupling agent, for masking the carboxylic acid functional groups of the protein and/or polysaccharide. The coupling agent is used to activate the carboxylic acid functional groups of the protein and/or polysaccharide by forming an activated ester. These activated esters will then react with nucleophiles selected from the group consisting of 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 the polysaccharide) or in the culture medium, as is the case with nucleophilic buffers, for example Tris.

The coupling agent may be chosen, for example, from carbodiimides, among which 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), Diisopropylcarbodiimide (DIC) and Dicyclohexylcarbodiimide (DCC) may be cited. The carbodiimide may be used in combination with other reagents, such as N-hydroxysuccinimide and triazole, among which 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) may be cited.

The masking agent may also be difunctional or polyfunctional in order to establish a bridge between reactive residues of the protein (bridging compounds, such as bisaldehydes, bis-N-hydroxysuccinimide esters, bisacylchlorides and derivatives thereof).

For example, an acylating agent, such as acetic anhydride, may be used at a concentration of 0.01M to 1.8M, such as 0.1M to 1.0M, for example 0.2 to 0.6M. The concentration of masking agent used to treat the sample may be adjusted depending on the concentration of protein and/or polysaccharide in the sample, the masking agent used, the source of the sample, and the lysis conditions used. The concentration of the acylating agent may be chosen such that it reacts with the amine and/or carboxylic acid functional groups of the protein and/or polysaccharide and not with the nucleic acid.

In the presence of the masking agent, the incubation time of the lysed sample when the lysate or lysis and treatment steps are performed simultaneously is typically between 1 second and 30 minutes, but preferably less than 10 minutes.

In one embodiment, the step of controlling the acidity of the treated lysate is performed, for example, with sodium hydroxide (NaOH), in particular in order to obtain a treated lysate with a pH preferably between 5 and 7. The amount of sodium hydroxide added in this step is low, so this step can only control the pH before the capture step, rather than a step of deprotecting the masked amine and/or carboxylic acid functional groups of the protein and/or polysaccharide. The step of controlling the acidity of the treated lysate is optional; in a particular embodiment, this step is absent.

At the end of the treatment step, a treated lysate comprising modified proteins and/or polysaccharides is obtained. In particular, at least a portion of the amine and/or carboxylic acid functional groups of the protein and/or polysaccharide are masked after treatment; preferably, the nucleic acid is not modified by the masking agent.

Capture

The capture step comprises placing the treated lysate in the presence of a solid support, for example a silica-based solid support, under conditions allowing the adsorption of the nucleic acids (solid phase extraction of the nucleic acids). Silica-based supports, such as silica beads or particles, allow the separation of nucleic acids retained by adsorption on silica from other cellular contaminants (membranes, proteins treated with masking agents, etc.). Advantageously, no antibody is used in the capture step.

In a specific embodiment, the step of treating the sample or lysate with a masking agent is directly followed by a capture step. In this case, there is no intermediate step between the masking step and the capturing step. In particular, there is no step of deprotecting the masked amine and/or carboxylic acid functional groups between the masking step and the capture step or during the capture step. Preferably, during the capturing step, the sample comprises modified proteins and/or polysaccharides (i.e. masked by the masking agent) and unmodified nucleic acids (i.e. unmasked by the masking agent).

In a specific embodiment, a sufficient amount of silica beads or particles are added to the treated lysate. In this embodiment with silica beads, preferably, the conditions capable of adsorbing nucleic acids comprise the presence of a chaotropic agent, for example at a pH of 4 to 8, for example guanidine hydrochloride or guanidine thiocyanate in a buffer medium with the aid of an organic compound, for example a salt of TRIS (TRIS (hydroxyethyl) aminomethane), acetate, phosphate, citrate or MES (morpholinoethanesulfonic acid).

Detergents, preferably Triton X100 or one of its analogues (Tergitol, Tween, Brij, Nonidet, Ecosurf, etc.) may also be added. In another embodiment, the detergent is contained in a lysis buffer. The silica-based support is preferably negatively charged at pH 5.0 to pH 8.0.

In a particular embodiment, no ionophore or alcohol is used during the trapping step by adsorption on the solid support. Suitable carriers, in particular amino carriers, will then be used.

As examples of silica beads or particles that can be used in the capture step, particles contained in the following kits can be cited: NucliSENS easy MAG Magnetic Silica (BioMerieux), Film Array (BioMerieux/Biofire Diagnostic), Qiasmphony Kits (Qiagen) and Magnapure Kits (Roche).

In another embodiment, the silica-based solid support is an extraction column with a silica membrane. For example, kits sold under the names of Purelink Genomic DNA Extraction Kit (Invitrogen) and DNeasy Bloodsand Tissue Kit (Quiagen) can be cited. The treated lysate was precipitated on the column with a silica membrane and then centrifuged to pass the lysate through the column. The nucleic acid is retained on the column and proteins, polysaccharides and other cell debris pass through the column.

Other silica-based supports and their use in nucleic acid extraction are described in particular in the following articles: cade et al, Nucleic acid purification using micro-fabricated silicon structures, biosensors and Bioelectronics 19,59-66 (2003); melzak, c.s.sherwood, r.f.b.tube, c.a.haynes, Driving Forces for DNA administration into a silicon in per solution 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 materials in a Miniaturized Format, Analytical biochemistry283,175-191 (2000); wolfe et al, aware a microchip-based chromatography method for isolation of nucleic acids.Electrophoreses 23,727-733 (2002).

In another embodiment, magnetic silica particles are used. The magnetic silica particles may be retained by the magnet during the washing step and during a suitable elution step following capture. This embodiment is particularly preferred for automation of the extraction process. Examples of Magnetic particles that can be used include kits sold under the name NucliSENS easy MAG Magnetic silicon (BioMerieux).

In a particular embodiment, the method for extracting nucleic acids from an unfixed 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 it comprises a step of treating the sample with at least one reagent to mask the amine and/or carboxylic acid functional groups of the proteins and/or polysaccharides of the sample before the capturing step, and the capturing step is carried out directly after the step of treating the sample with the masking agent.

Washing and elution

After the capture step, one or more than one washing step may be performed in the presence of a washing buffer, thereby enabling the removal of contaminating elements without stripping the nucleic acids from the support. For example, the washing step may comprise a step of washing in a suitable washing buffer, such as a low salt solution, which may comprise an alcohol, and/or a step of washing in the presence of an alcohol to remove salts. In a preferred embodiment of the invention, at least one washing step is carried out.

If necessary, an elution step may then be performed. The elution step comprises releasing the nucleic acid retained on the solid support. Typically, buffers having a basic pH, e.g. pH 8 to 10, and a low ionic strength, e.g. Tris or borate buffers, are used. Alternatively, the detection or amplification step is performed directly after the capture and wash steps. The detection and/or amplification is carried out directly on a suitable solid support on which the purified nucleic acid is captured, without prior elution steps. Preferably this is done on beads or particles or membranes, more preferably on particles which may or may not be magnetic.

The nucleic acids purified by the above-described method (with or without an elution step) can be used in particular for amplification and in vitro detection.

Additional steps of amplification and/or detection

The invention is particularly suitable for preparing nucleic acids for use (in an in vitro detection assay) in the detection of target nucleic acids in a sample, in particular after amplification of the target nucleic acids.

Accordingly, the present invention relates to a method for detecting a target nucleic acid sequence in a biological sample, said method comprising:

(i) extracting nucleic acid from a biological sample that may contain a nucleic acid sequence of interest,

(ii) detecting the nucleic acid sequence in the extracted nucleic acid.

According to a particular embodiment, the detection step (ii) comprises a step of amplifying the target nucleic acid sequence.

Accordingly, the present invention also relates to a method for amplifying a nucleic acid, comprising: (i) performing the method of extracting nucleic acid on a suitable vector as described above, and (ii) a nucleic acid amplification step using a DNA polymerase.

One advantage of the present method is that the amplification step can be performed immediately after the capture step. Because the nucleic acid is not modified by the masking agent, it is not necessary to deprotect the nucleic acid prior to amplification.

In a specific embodiment, the amplification method is performed using a DNA polymerase, such as Taq polymerase, Pfu polymerase, T7 polymerase, Klenow fragment of e.

In another embodiment, which can be combined with the preceding embodiments, the amplification method is Polymerase Chain Reaction (PCR) amplification, which is well known to those skilled in the art. The PCR protocol comprises 20 to 40 cycles, for example each cycle comprising at least (i) a stage of denaturing the DNA to be amplified at a temperature typically between 90 ℃ and 95 ℃, (ii) a stage of hybridizing the primers with the DNA to be amplified at a temperature typically between 55 ℃ and 65 ℃, and (iii) an extension stage at a temperature typically between 68 ℃ and 75 ℃.

Variants of the nucleic acid amplification method by PCR may also be performed. In particular, nested PCR, quantitative PCR (or qPCR), semi-quantitative or real-time PCR, error-prone PCR or reverse transcription PCR (RT-PCR) may be cited. Other amplification techniques, such as LAMP, NASBA, TMA, RPA, LCR, RCR, 3SR, RCA, SDA, or any nucleic acid amplification technique known to those skilled in the art, may also be performed.

Nucleic acid extraction kit

The invention also relates to a nucleic acid extraction kit or kit comprising at least:

i. the agent for masking amine and/or carboxylic acid functions of proteins and/or polysaccharides as described hereinbefore, for example an acylating agent, preferably acetic anhydride or a dry form thereof, N-hydroxysuccinimide acetate,

a solid support suitable for extracting nucleic acids,

a catalyst for the reaction of masking amine and/or carboxylic acid functional groups with a reagent, where appropriate, and/or

Where appropriate, a coupling agent.

The extraction kit may further comprise buffers, control elements and/or instructions for use.

In a particular embodiment, the extraction kit does not comprise reagents to deprotect the masked amine and/or carboxylic acid functional groups.

In a particular embodiment, the extraction kit does not comprise an organic solvent.

In another specific embodiment, the kit may comprise the following elements:

i. the coupling agent, such as EDC,

a silica-based solid support suitable for extracting nucleic acids, such as silica beads, in particular magnetic silica beads, and at least one of the following optional elements:

lysis buffer comprising Tris or other nucleophilic buffer,

one or more than one wash buffer, and/or

v. optionally an elution buffer,

an optional catalyst.

In a particular embodiment, the kit comprises a column with a silica membrane as a silica-based solid support.

These kits are particularly suitable for carrying out the extraction methods described hereinbefore. The method according to the present disclosure will be better understood with the aid of the embodiments detailed below and with reference to the accompanying drawings.

Drawings

FIG. 1: DNA capture on solid supports is inhibited by certain proteins under discrete conditions.

FIG. 2: DNA capture on solid supports is inhibited by certain polysaccharides under discrete conditions.

FIG. 3: DNA capture on solid supports was inhibited by model monofunctional polymers.

FIG. 4: A. nucleic acids contained in blood samples were extracted using the easyMAG extraction kit (biomerriux) and acetic anhydride (Ac) ₂O) or acetic acid (AcOH) for pretreatment of the lysate (capture in 3M GuHCl + 1% Triton and washing with ethanol). B. The amount of nucleic acid was measured by chromatographic analysis.

FIG. 5: HPLC analysis of the extract eluate of the DNA and RNA mixture, with or without acetic anhydride treatment. The upper diagram: eluate obtained from a mixture of extracted 10. mu.g DNA and RNA and 5.2mg protein in the presence of acetic anhydride. The following figures: eluate obtained from a mixture of extracted 10. mu.g DNA and RNA.

FIG. 6: fluorescence detection (RFU) curve of CMV viral nucleic acid in blood amplified by PCR in real time. The curves of fig. 6 correspond to control amplifications performed after extraction without biomatrix (triangles), without the biomatrix treated with acetic anhydride (X-shape) and with the biomatrix treated with acetic anhydride (circle), respectively.

FIG. 7: with or without acetic anhydride Ac prior to extraction₂Sensitivity (Ct) for CMV virus detection in O-treated blood. Left: 10e4 copies of CMV in Tris HCl; the method comprises the following steps: 10e4 copies of CMV in blood; and (3) right: blood + Ac₂10e4 copies of CMV in O.

FIG. 8: in the presence of a mixture of human serum albumin and hemoglobin, with or without Ac₂In the case of O treatment, DNA was extracted on a column having a silica membrane. Left (dark gray): without Ac ₂O treatment; right (light gray): with Ac₂And (4) O treatment.

FIG. 9: chromatograms corresponding to the intact oligonucleotide (a), the same oligonucleotide (B) in the presence of nuclease P1 and thus degraded, and the same oligonucleotide (C) in the presence of nuclease P1 and acetic anhydride showing better oligonucleotide stability due to acylation of the nuclease.

Examples

General conditions

The general analytical conditions for the compounds used in the following examples are described below:

LC-MS analysis was performed using a WATERS Alliance 2795HPLC system equipped with a PDA 996 photodiode array detector (Waters), a ZQ 2000 mass spectrometer detector (Waters) and Empower software version 2. The ZQ 2000 mass spectrometer has an electrospray ionization source. Ionization was performed in positive ion mode with a cone voltage of 20V and a capillary voltage of 3.5 kV.

Conditions used for HPLC analysis were as follows (conditions a and B):