DNA purification method by double lysis

Abstract

The invention falls within the field of infectious disease diagnosis and relates to a novel method for purifying DNA from microorganisms of interest present in a biological sample, comprising at least one double differential lysis, in order to improve the isolation and detection of DNA from microorganisms of interest. This results in an improvement in the detection limit of said purification and isolation method.

Description

Title of the invention: Method for DNA purification by double lysis. Technical field.

[0001] The invention relates to the field of infectious disease diagnosis, particularly emergency diagnosis. The invention thus concerns a new method for isolating and / or purifying DNA from microorganisms of interest in a biological sample, said sample preferably being a biological sample of animal origin, particularly human origin. In particular, the method comprises at least one differential double lysis step, in order to improve the diagnostic performance, especially the limit of detection and the sensitivity, of such an isolation and / or purification method. State of the art

[0002] In the field of infectious disease diagnosis, numerous diagnostic methods and kits implementing them have been developed. Serological, microbiological, and molecular biology methods are well-known. Molecular biology methods such as nucleic acid amplification (particularly Polymerase Chain Reaction, or PCR) have led to significant progress in the diagnosis of infectious diseases. Indeed, these methods are rapid, sensitive, and specific. However, their sensitivity and specificity may be incompatible with routine or clinical use, especially for emergency diagnosis. Among these drawbacks is the difficulty of processing excessively large sample volumes within a limited timeframe.

[0003] In the context of diagnosing infectious diseases, particularly in emergency situations, it is advantageous to use biological samples such as, but not limited to, blood, cerebrospinal fluid, bronchoalveolar lavage fluid, urine, biopsies, or stool. These samples all have in common that they contain numerous biological elements in large quantities, such as cells, proteins, and microorganisms, particularly bacteria.

[0004] However, the number of microbial cells to be sought for the diagnosis of certain infectious diseases may be extremely small. For example, one bacterial cell in 10 mL of sample. The development of diagnostic tests to detect the presence of these bacteria in very small quantities therefore requires the use of large sample volumes, on the order of 10 mL or further, in order to increase the statistical chances of detecting and identifying the pathogen. In this context, the aforementioned samples are of particular interest.

[0005] To develop such diagnostic methods, saponin is commonly used. Naturally produced by plants or animals, saponin is a molecule with detergent properties, capable of inducing differential lysis of animal cells, particularly human cells, unlike microbial cells. In particular, saponin is selective for cells containing cholesterol, which is a component and stabilizes the cell membrane, especially the membrane of animal cells. Conversely, Gram-positive and Gram-negative bacterial cells do not contain it.This property is therefore of particular interest for the development of diagnostic tests because it allows for the selective elimination of animal cells without damaging the bacterial cells of interest.

[0006] Furthermore, differential lysis with a saponin solution is very effective on small volumes of treated biological samples, unlike large volumes of biological samples. In the latter case, efficiency is reduced and incomplete, leading to a significant accumulation of animal DNA during the extraction processes. However, the accumulation of animal DNA has the disadvantage of competing with the microbial DNA of interest during the DNA purification steps of the biological sample. As a result, the detection limit of diagnostic tests is degraded when animal DNA is incompletely removed. Yet, the detection limit of such a kit is an important parameter when the target bacterium is present in very small quantities in the biological sample.

[0007] There is therefore a need to develop a new method for diagnosing infectious diseases based on molecular biology techniques, particularly PCR, that overcomes the aforementioned drawbacks. Specifically, it is necessary to address the disadvantages associated with using molecular biology techniques with large sample volumes combined with the detection and identification of a bacterium of interest present in very small quantities.

[0008] To meet this need, the invention proposes a method for isolating and / or purifying DNA from microorganisms of interest present in a biological sample, said method comprising two successive differential lysis steps, which makes it possible to lyse and eliminate animal cells, in particular human cells, very efficiently.

[0009] Furthermore, the removal of animal cells and their DNA prevents the clogging of silica columns by animal DNA, thus improving the recovery of microbial DNA from the silica surface. The removal of animal DNA also significantly reduces the saturation of silica surfaces used for purification (e.g., silica columns or beads). silica magnets). However, this prevents small quantities of microbial DNA from binding. As a result, column saturation leads to a very poor recovery rate of this microbial DNA of interest, contrary to the objective of the present invention.

[0010] Silica surfaces, in the form of columns or beads, are very commonly used in DNA purification processes for the purpose of using this DNA with conventional molecular biology techniques such as PCR, qPCR, or sequencing. Clogging or saturation of said silica surfaces inevitably leads to a drastic decrease in the detection limit of the method. Conversely, the method according to the present invention increases the detection limit. Summary of the invention

[0011] Thus, the invention relates to a method for isolating and / or purifying DNA from microorganisms of interest present in a biological sample; said method comprises the following steps: a. to bring a biological sample into contact with a saponin solution, in order to lyse the animal cells, including human cells, contained in said biological sample, b. Centrifuge the mixture from step a), discard the supernatant, and retain the pellet containing the cells of microorganisms of interest. c. bring the pellet into contact with a saponin solution in order to lyse any animal or human cells not lysed in step a), d. eliminate animal or human DNA by treatment with at least one nuclease, e. inactivate the nuclease, by any suitable means, in order to be able to lyse the microbial cells.

[0012] Preferably, the volume of the biological sample is between 0.1 mL and 40 mL, more preferably between 5 mL and 20 mL, and even more preferably between 8 mL and 10 mL. Such a volume is particularly useful for isolating microorganisms present in very small quantities in the sample.

[0013] In the context of the invention, the biological sample is a sample comprising animal cells, advantageously human cells.

[0014] According to another preferred object, during step a), the ratio between the volume of biological sample and the volume of saponin solution is between 0.5:1 and 2:1, most preferably the ratio is 1.125:1.

[0015] According to another preferred object, during step c), the ratio between the volume of the pellet and the volume of the saponin solution is between 1:1 and 1:5, most preferably the ratio is between 1:1 and 1:2.

[0016] According to a preferred object of the invention, the biological sample is a fresh biological sample of animal origin, in particular of human origin, said biological sample is very preferably chosen from blood, cerebrospinal fluid, bronchoalveolar lavages, urine, stool, biopsies and their mixture, very preferably, this is a whole blood sample.

[0017] According to another preferred object of the present invention, the method comprises a step d) of removing the animal DNA present in the mixture recovered in step c), said step d) comprising the addition of nucleases in an appropriate quantity to remove the animal DNA present in the mixture, the quantity of nucleases being very preferably between 1*10A3 and 2*10A6 units (U) per biological sample, in particular the volume of said biological sample is between 0.1 and 40mL.

[0018] According to another object of the present invention, the method comprises a step e) of inactivating the nucleases added in step d), said inactivation step e) being carried out, preferably, by the addition of a divalent ion chelator (e.g., EDTA, EGTA, etc.), and / or the addition of guanidine, and / or heating the sample, and / or digesting the nucleases with proteases. Step e) being carried out most preferably by the addition of EDTA at a final concentration between 0.1 mM and 1000 mM, and even more preferably between 100 mM and 500 mM.

[0019] According to another object, the method further comprises a step of lysis of the microbial cells present in the mixture after step e) of nuclease inactivation. This can be carried out by mechanical action with beads in the presence or absence of lysis enzymes, or by the action of lysis enzymes alone, or by the action of a hydroxide solution, or by the action of guanidine.

[0020] Preferably, the beads used for mechanical lysis have a specific diameter that facilitates liquid recovery by pipetting and allows the present method to be implemented in a way that is compatible with automation. Thus, the diameter of the beads used for mechanical lysis is most preferably between 1 mm and 6 mm.

[0021] In order to optimize and further improve the detection limit of the present method, the saponin solution is preferably in fresh or frozen form. When in frozen form, the saponin has previously been preserved by freezing, more preferably at a temperature of at least -20°C.

[0022] According to another preferred object, the method includes an additional step of removing polypeptides present in the biological sample; this step includes the addition of a heat-resistant protease followed by heat treatment. This step is implemented after the step of removing animal DNA by the Nuclease treatment and its inactivation. Indeed, protease can destroy or damage the bacterial cell wall, thereby releasing the microbial DNA of interest into the mixture. Protease thus reduces competition between proteins and DNA during their binding to silica surfaces. Furthermore, it eliminates PCR inhibitors such as hemoglobin. Therefore, in the context of the present invention, nuclease treatment can only be carried out before protease treatment.

[0023] Advantageously, the quantity of protease is between 10 units and 10A6 units per sample.

[0024] According to another preferred embodiment, the method includes an additional step of adding a reducing agent capable of reducing the disulfide bonds of proteins in order to improve the digestion and elimination of polypeptides present in the biological sample. This step is most preferably carried out before, during, or after the addition of said protease during the elimination of polypeptides present in the biological sample. By "before or after" the addition of said protease, for the purposes of the invention, is meant the step necessarily juxtaposed with that of adding a protease, whether it is pre- or post-addition of said protease.

[0025] Very preferentially, the heat-resistant protease is papain.

[0026] According to another preferred object, the heat treatment temperature is between 10°C and 100°C, more preferably 80°C. The duration of said polypeptide removal step can be between 1 and 30 minutes.

[0027] According to another preferred aspect of the invention, the method includes a final additional step of purifying the DNA of microorganisms by means of a heat treatment of said DNA from the purified microorganism. This step is necessarily the last step of the method for purifying and isolating microbial DNA. Indeed, it aims to eliminate all traces of alcohol (such as isopropanol or ethanol) and thus allow the use of the entire sample so as not to restrict the maximum detection capacity and therefore to obtain the best detection limit.

[0028] This final purification step is carried out by heat treatment, the temperature of said heat treatment being preferably between 60°C and 100°C, more preferably 90°C. Even more preferably, the duration of said heat treatment is between 1 and 30 min.

[0029] Advantageously, the method according to the present invention thus makes it possible to obtain a detection limit of 20 total CFU, more preferably 10 total CFU. By way of example, for 8 mL of adult blood, the limit is between 1.2 and 2.5 CFU / mL of biological sample.

[0030] Other features and advantages will become apparent from the detailed description of the invention, the examples and figures that will follow. Brief description of the Figures

[0031] [Fig-1] represents the results of the double lysis performed on a volume of blood Fresh 9 mL samples were compared to those treated with single lysis. Columns with blood treated by single lysis clogged (Panel A), with only a small portion of the lysate passing through. To recover sufficient lysate for the subsequent steps, these columns were centrifuged at high speed to force the lysate through. Bacteria present in the blood samples were not detected by qPCR when the blood was treated by single lysis (Panel B; squares forming a horizontal line), but they were readily detected when the blood was treated by double lysis (Panel B; curve with open circles). Finally, the quantification of human cell DNA detected by qPCR showed that a greater quantity of human DNA was present in the silica-column-purified samples after single lysis (Panel C; line with open squares) than after double lysis (Panel C; curve with open circles).

[0032] [Fig. 2] shows the results of treating blood cell DNA with different high amounts of nuclease for different times. Human cell DNA is purified on a silica column after treatment for 15 minutes (panel A; curves with open squares) or 120 minutes (panel B; curves with open circles) with a high amount of nuclease (5000 units). The results demonstrate that extending the incubation time removes a greater amount of human DNA. Panel B: Human cell DNA is purified on a silica column after treatment for 15 minutes with a high amount of nuclease (5000 units) (panel B; curves with open squares) or after treatment for 15 minutes with a high amount of nuclease (150,000 units) (panel B; open circles forming a horizontal line).The results demonstrate that greatly increasing the amount of nucleases in the reaction allows for the complete elimination of human DNA.

[0033] [Fig. 3] shows the lysis of Gram-positive Staphylococcus aureus bacterial cells by glass beads with a diameter of 1 mm or 3 mm compared to a reference enzymatic method using lysostaphin. Staphylococcus aureus bacterial cells inoculated into whole blood are lysed by an enzymatic method by incubation in the presence of a Staphylococcus aureus-specific lysis enzyme, lysostaphin (open and solid circles), or by a mechanical method with glass beads with a diameter of 1 mm (open and solid squares) or 3 mm (open and solid triangles). The bacterial DNA is then purified and detected by a Staphylococcus aureus-specific qPCR. The three lysis methods tested allow the detection of Staphylococcus aureus at comparable Cq values. The use of glass beads with a diameter of 1 mm or 3 mm therefore allows a lysis of the cells of a Gram-positive bacterium, Staphylococcus aureus, comparable to an enzymatic lysis specific to this bacterium.

[0034] [Fig. 4] shows the results of freezing a saponin solution. The whole blood pellet after lysis of blood cells by a saponin solution stored for 1 hour at room temperature (A), 20 days at 4°C (B), or 20 days at -20°C (C). Only the saponin solution stored at -20°C for 20 days produces a pellet of the same small volume as the saponin solution stored at room temperature for 1 hour, indicating that the detergent properties of the saponin solution stored at -20°C are retained and therefore that the blood cells are effectively removed.

[0035] [Fig. 5] shows the results of polypeptide removal by heat treatment with an unusually high amount of heat-resistant protease in the presence of DTT at a final concentration of 8.3 mM. SDS-PAGE gel of whole blood samples (0.2 µl) after first lysis with a saponin solution (A), after double lysis with a saponin solution (B), and after treatment with 60,000 units of papain for 10 minutes at 80°C (C). The papain treatment removes all polypeptides present in the biological sample after only 10 minutes of treatment. Detailed description of the invention

[0036] Definition

[0037] By "microorganism" or "microbe", in the sense of the invention, means bacteria, viruses, yeasts, which one seeks to isolate and identify in order to identify and / or confirm the disease in an individual.

[0038] For the purposes of this invention, "animal" means any living being classified as "animal" in the "scientific classification of species" (also called "biological classification"). Thus, "animal" includes mammals, in particular human beings.

[0039] For the purposes of this invention, "total CFU" means the total number of CFUs, CFU being the English term for Colony Forming Unit. A CFU corresponds to a very small cluster of cells whose growth on a solid medium leads to the formation of a colony. In its most rudimentary form, a CFU may correspond to only a single microbial cell.

[0040] For the purposes of this invention, "fresh solution" or "fresh saponin solution" means a reagent solution that has been prepared a few minutes to one hour before use and stored at room temperature before use.

[0041] For the purposes of this invention, "frozen solution" means a fresh saponin solution that has been preserved by freezing, preferably at a temperature of at least -20°C.

[0042] Method according to the invention

[0043] In the context of the present invention, efficient differential lysis is essential for the development of a diagnostic test. However, in some cases, the microorganism of interest may be present in the biological sample in very small quantities. This necessitates the use of large volumes of biological sample, which are incompatible with differential lysis, a technique that is highly effective on small volumes. The inventors have therefore developed a new method comprising two successive differential lyses, overcoming the drawbacks of the prior art.

[0044] The present invention therefore relates to a method for isolating and purifying DNA from microorganisms of interest present in a biological sample, said method comprising the following steps: a. to bring a biological sample into contact with a saponin solution, in order to lyse the animal cells contained in said biological sample, b. Centrifuge the mixture from step a), discard the supernatant, and retain the pellet containing the cells of microorganisms of interest. c. bring the pellet into contact with a saponin solution in order to lyse the animal cells not lysed in step a), d. Eliminate animal DNA by nuclease treatment, e. Inactivate the nuclease.

[0045] Successive double lysis thus avoids several drawbacks associated with using a large sample volume, notably the clogging and saturation of silica surfaces (such as silica columns or beads) by blood cell DNA. These silica surfaces, such as columns or beads, are commonly used in DNA purification processes. Consequently, the method according to the invention improves the recovery of microbial DNA (or microorganisms) of interest from the biological sample onto a silica surface.

[0046] Preferably, the volume of the biological sample is between 0.1 mL and 40 mL, more preferably between 5 mL and 20 mL, and even more preferably between 8 mL and 10 mL. Such a volume is particularly important for being able to isolate a microorganism of interest present in very small quantities, as low as 1 or 2 bacteria per milliliter (mL), more preferably between 1 bacterium per milliliter and 500 bacteria per milliliter.

[0047] According to another preferred object, during step a), the ratio between the volume of biological sample and the volume of saponin solution is between 0.5:1 and 2:1, most preferably the ratio is 1.125:1.

[0048] On the other hand, the biological sample is preferably a biological sample of animal origin, comprising animal cells, in particular human cells. According to another preferred specification, the sample is a fresh biological sample selected from blood, cerebrospinal fluid, bronchoalveolar lavage fluid, urine, feces, biopsies, and mixtures thereof. Even more preferably, the biological sample is a whole blood sample.

[0049] According to a variant of the invention, the method for isolating and purifying DNA from microorganisms of interest present in a biological sample, said method comprises the following steps: a. to bring a liquid biological sample into contact with a saponin solution, in order to lyse the animal cells contained in said biological sample, b. Centrifuge the mixture from step a), discard the supernatant, and retain the pellet containing the cells of microorganisms of interest. c. bring the pellet into contact with a saponin solution in order to lyse the animal cells not lysed in step a), d. culture the mixture from step c) comprising the microbial cells, for example on a Petri dish.

[0050] Once the animal cells have been lysed and removed, the method includes a step of adding nucleases to remove the animal DNA. It is well known to those skilled in the art to implement such a step to remove animal DNA using any enzyme exhibiting nuclease activity. However, the quantity of enzymes typically added, on the order of a few units per reaction / sample, is insufficient for effective removal in a short time for a diagnostic test, particularly an emergency one. Indeed, the amount of animal DNA will be all the more excessive as the volume of biological sample to be processed increases, which is the case here. For example, a diagnostic test using a large volume of blood, on the order of 10 mL, must be able to handle an amount of animal DNA of approximately 70 pg, or about 100,000 genome copies.However, these 70pg of animal DNA will have the disadvantage of clogging or saturating the silica surfaces, negatively impacting the detection limit of diagnostic tests. Yet, as described previously, the quantity of microbial DNA of interest is very small, from 5 to 500 fg, or 1 to 100 copies of the bacterial genome.

[0051] Also, in order to overcome this drawback, related to the volume of sample to be processed in a short time, the inventors added a very high quantity of nuclease allowing This reduces the time required to eliminate animal DNA to just a few minutes. The combination of the various steps constituting the present method according to the invention thus allows the destruction of animal DNA without eliminating the microbial DNA of interest, when the amount of nuclease is very high.

[0052] Also, according to a particular object of the invention, the method according to the invention also includes a step of removing animal DNA present in the mixture of step c), by means of nucleases, the quantity of nucleases being preferably between 1*10A3 and 2*10A6 units (U) per biological sample.

[0053] The nuclease can also be inactivated by adding a divalent ion chelator such as EDTA or EGTA in order to inactivate the nuclease used to remove animal DNA, before the lysis step of the target microorganisms.

[0054] In order to further improve the detection limit of the method according to the present invention, and thus reconcile the processing of a large volume while maintaining good sensitivity and specificity of the test, the method according to the present invention preferably comprises an additional step of lysis of microorganism cells after the step of removal of animal DNA and inactivation of the nuclease. This lysis step is preferably carried out by mechanical action and / or by enzymatic action and / or by chemical action.

[0055] Most preferably, the mechanical action is carried out by means of beads, with or without lysis enzymes. According to another preferred embodiment, the enzymatic action is carried out by means of suitable enzymes known to those skilled in the art. Finally, according to yet another preferred embodiment, the chemical action is carried out by means of the addition of a hydroxide or guanidine solution.

[0056] When the mechanical action is performed using balls, said balls preferably have an unusually large diameter to facilitate the recovery of liquids by pipetting, reduce the time required for sample processing, and make this step compatible with automation. Also, more preferably, these balls have an average diameter of between 1 mm and 6 mm.

[0057] On the other hand, according to another preferred object of the invention, the saponin solution is a fresh solution or one preserved by freezing, preferably at a temperature of at least -20°C. In other words, the saponin solution used in steps a) and c) to lyse the animal cells included in the biological sample was kept at -20°C, then thawed and brought into contact with the sample at room temperature.

[0058] Indeed, saponin solutions stored at room temperature or 4°C lose their detergent properties within a few days and are no longer able to lyse animal cells optimally. To guarantee the preservation of the lysis properties of a saponin solution over time, and therefore its maximum effectiveness, the The inventors kept said solution at a temperature of at least 20°C. This unusual temperature ensured the stability of the detergent properties of the saponin solution, further improving the effectiveness of the method according to the invention.

[0059] In the context of the invention, another drawback associated with using a large sample volume is the excess of polypeptides from animal cells, which can clog or saturate silica surfaces and thus prevent the binding of microbial DNA present in the sample. Furthermore, certain polypeptides from biological samples are known to inhibit PCR reactions, potentially preventing the detection of the DNA of interest by this method even when present in very small quantities. These polypeptides can persist in the sample despite the purification of the DNA of interest on a silica surface, and it may be necessary to remove them before this purification step. Adding proteases is known to overcome these drawbacks.However, the quantity of proteases typically supplied is insufficient to eliminate all polypeptides present in a large biological sample within a short timeframe. This significantly reduces the detection limit of the method, where the DNA of microorganisms of interest may, in some cases, be present in very small quantities in the sample (5 to 500 fg, or 1 to 100 copies of bacterial genome). Furthermore, only a small amount of the supplied protease is actually active. For example, Proteinase K has a low activity per unit mass, on the order of approximately 1 to 50 units / mg.

[0060] Furthermore, the inability of the proteases used to efficiently remove polypeptides from the biological sample reduces the capacity of microbial DNA to be captured by the silica surfaces used for DNA extraction and increases the amount of PCR inhibitors in the sample. Consequently, the detection limit of the test will again be negatively impacted by the residual presence of polypeptides in the sample.

[0061] To further improve the method according to the present invention, the inventors have therefore added an additional step consisting of adding at least one heat-resistant protease in a very high quantity. Advantageously, the quantity of protease is between 10 units and 10⁶ units per sample.

[0062] Thus, according to another object, the method comprises an additional step of removing polypeptides present in the biological sample; said polypeptide removal step comprising the addition of at least one heat-resistant protease combined with heat treatment. More preferably, the heat-resistant protease is papain. This advantageously exhibits a specific activity in the range of 10,000 to 50,000 units / mg.

[0063] Furthermore, coupling at least one heat-resistant protease with a high incubation temperature significantly increases the enzyme's efficiency, accelerates polypeptide removal, and achieves near-total polypeptide elimination. This results in excellent polypeptide removal during DNA extraction processes, leading to very low detection limits for the target DNA and therefore greater sensitivity.

[0064] Preferably, the heat treatment temperature is between 10°C and 100°C, more preferably 80°C.

[0065] Most preferably, the polypeptide removal step includes the addition of papain combined with a heat treatment between 10°C and 100°C, more preferably 80°C.

[0066] Furthermore, the removal of certain proteins may require the reduction of disulfide bonds that help maintain their three-dimensional structures and therefore their functions, and can block DNA capture on silica surfaces or inhibit the amplification of nucleic acid targets during PCR. Thus, the present method advantageously includes the addition of at least one reducing agent during the polypeptide removal step.

[0067] Thus, according to a preferred object, at least one reducing agent is used, this being chosen from DTT, DTE, Beta-mercaptoethanol, TCEP, glutathione, and Thioglycerol, at a concentration between 0.1 mM and 100 mM.

[0068] Finally, the method advantageously includes an additional final step of purifying the DNA from microorganisms after purification on silica. This final step aims to further improve sensitivity by means of heat treatment of the purified microbial DNA. Preferably, the temperature of the heat treatment of the eluate containing the purified DNA is between 60°C and 100°C, preferably 90°C, for a duration of between 5 and 30 minutes. This allows for the elimination of residual alcohol (isopropanol or ethanol) commonly used by those skilled in the art during such a DNA purification process. By eliminating the residual alcohol, the amount of microbial DNA in the eluate that can be used is maximized, thus obtaining a better limit of detection.

[0069] According to another object of the invention, steps a) and b) are repeated at least once before treatment with nucleases, preferably at least twice.

[0070] According to another object of the invention, after step c., the method does not include a centrifugation step in order not to dilute the sample too much.

[0071] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results. Examples

[0072] Example 1: Comparison of single and double lysis methods.

[0073] The purpose of this test is to demonstrate the improved efficiency of the method according to the present invention compared to the prior art.

[0074] The test protocol is as follows. From 9 mL samples of human whole blood containing between 9 and 24 bacterial cells, said samples are mixed with 8 mL of saponin and then centrifuged for 20 minutes at 24,000 g. After removal of the supernatant, the two pellets resulting from single lysis are directly treated with a nuclease and a protease to remove excess human DNA and polypeptides. The samples are then loaded onto silica columns, and the DNA is purified according to the recommendations of the Macherey-Nagel Blood L Kit. To obtain pellets that have undergone double lysis, the pellets resulting from single lysis are again mixed with 400 pL of saponin to lyse the remaining human cells before being treated according to the same protocol as the single-lysis pellets.

[0075] The inventors then observed that the columns of samples treated by single lysis were clogged and that the sample could only partially pass through the column, unlike samples treated by double lysis ([Fig. 1], Panel A). In order to recover sufficient lysate for further analysis, the samples were centrifuged at very high speed to optimize the passage of the lysate through the clogged columns. The samples were then purified and analyzed by qPCR to measure the recovery rate of microbial DNA present in the initial biological sample, and to measure the amount of human cell DNA co-purified during passage through the silica column.

[0076] The target bacteria present in whole blood are readily detected when samples are treated by double lysis, but are not detected when samples are treated by single lysis (see [Fig. 1], panel B and Table 1 below; N / A: not detected). The remaining amount of DNA from human cells that is to be eliminated is lower for samples treated with double lysis compared to those treated by single lysis ([Fig. 1], panel C and Table 1).

[0077] In this example, the improvement in the removal of human DNA provided by the double lysis method is approximately a factor of 64. The double lysis method of the samples therefore makes it possible to improve the detection of the bacterial targets sought and a better removal of DNA from human cells.

[0078] [Tables] Type of lysis Cq Klebsiella pneumoniae Cq DNA blood cells s Simple lysis #1 N / A 21.13 Simple lysis #2 N / A 20.11 Double lysis #3 35.14 26.26 Double lysis #4 36.98 25.03

[0079] Example 2 - Rapid removal of DNA from human cells by a high amount of nuclease

[0080] The aim of this test is to demonstrate the improved efficiency of the method according to the present invention when it includes a step of adding nucleases in a high quantity.

[0081] The test protocol is as follows. Each 9 mL sample of whole blood is mixed with 8 mL of saponin solution and then centrifuged for 20 minutes at 24,000 g. The supernatant is discarded, and the pellets are treated again with 400 pL of saponin solution. These samples are then treated with a nuclease to remove excess human DNA and with a protease to remove human polypeptides, and the microbial DNA is purified on silica columns. The amount of human cell DNA purified on the silica columns is measured under different conditions. The samples were treated with a high amount of nucleases (5,000 units) for 15 minutes or 120 minutes before silica column purification. The results are shown in [Fig. 2], panel A, and in Table 2 below.

[0082] [Tables2] Nuclease time Cq DNA blood cells 15 minutes #1 25.35 15 minutes #2 26.57 15 minutes #3 26.1 15 minutes #4 26.6 120 minutes #5 31.17 120 minutes #6 31.35 120 minutes #7 36.71 120 minutes #8 31.38

[0083] The inventors then observed that a 15-minute treatment with 5,000 units is insufficient to eliminate all human DNA. A longer incubation reduces the amount of DNA by several dozen times, but does not allow for its complete elimination. By significantly increasing the amount of nuclease used to treat the sample (150,000 units), it is possible to eliminate all DNA from blood cells in just 15 minutes, compared to the results obtained with a lower amount of nuclease (5,000 units). units). The results are presented in [Fig.2], panel B, as well as Table 3, below (N / D: not detected).

[0084] [Tables3] Nuclease quantity Cq DNA blood cells 5000 units #1 27.02 5000 units #2 32.48 150,000 units #3 N / A 150,000 units #4 N / A

[0085] Thus, the use of a very large quantity of nucleases (150,000 units) makes it possible to eliminate all of the DNA from human cells in just 15 minutes.

[0086] Example 3 - Lysis of bacterial cells by large diameter glass beads.

[0087] The aim of this test is to demonstrate the effectiveness of the method according to the present invention when it uses large diameter glass beads to lyse a Gram-positive bacterium compared to the use of a specific lysis enzyme for the Gram-positive bacterium Staphylococcus aureus.

[0088] The test protocol is as follows. A 9 mL sample of human whole blood containing between 31 and 115 Staphylococcus aureus bacterial cells is lysed with 8 mL of saponin solution and then centrifuged for 20 minutes at 24,000 g. After removal of the supernatant, the pellets are treated with 400 pL of saponin solution. After treatment with a nuclease, two samples are incubated with a Staphylococcus aureus-specific lysis enzyme, lysostaphin. The next two samples are then agitated in the presence of 1 mm diameter glass beads. The last two samples are agitated in the presence of 3 mm diameter glass beads. In the case of mechanical lysis with glass beads, the liquid is easily recovered by pipetting due to the large diameter of the beads and transferred to a new tube.All samples are then loaded onto silica columns and the DNA is purified according to the recommendations of the Macherey-Nagel Blood Kit L.

[0089] The samples are then purified and analyzed by qPCR to measure the recovery rate of microbial DNA released during the lysis of bacterial cells present in the starting biological sample.

[0090] The target bacteria are readily detected when a Staphylococcus aureus-specific lysis enzyme is used. They are also detected when large-diameter beads (1 mm and 3 mm) are used, and at a detection level comparable to the enzymatic method. The results are presented in Table 4 below and in [Fig. 3].

[0091] [Tables4] Treatment methods for Staphylococcus aureus: Lysostaphin #1 34.28, Lysostaphin #2 35.00, 1 mm beads #3 35.59, 1 mm beads #4 34.69, 3 mm beads #5 35.84, 3 mm beads #6 36.90

[0092] Thus, the present test demonstrates the effectiveness of the method according to the present invention when it uses large diameter glass beads, allowing in addition, simple recovery of the lysate compatible with automated pipetting processes while maintaining a lysis efficiency comparable to the reference enzymatic method.

[0093] Example 4 - Comparison of the effect of different storage temperatures on the preservation of the lysis properties of saponin over time.

[0094] The purpose of this test is to demonstrate the improved efficiency of the method according to the present invention when using frozen saponin.

[0095] The test protocol is as follows. 9 mL samples of whole blood are mixed with 8 mL of a saponin solution stored for 1 hour at room temperature (A), 20 days at 4°C (B), or 20 days at -20°C (C) and centrifuged for 20 minutes at 24,000 g. After removal of the supernatant, the pellet containing the unlysed whole cells is collected at the bottom of the tube.

[0096] The results are presented in [Fig.4].

[0097] Mixing whole blood with a saponin solution stored for only 1 hour at room temperature (A) or 20 days at -20°C (C) allows for complete removal of blood cells, leaving only a small volume of blood cells at the bottom of the tube thanks to the detergent properties of the saponin solution. Mixing whole blood with a saponin solution stored for 20 days at 4°C (B) does not allow for complete removal of blood cells, leaving a large volume of unlysed blood cells at the bottom of the tube because this saponin solution has lost its lysing properties. The pellet is different and consists of unlysed red blood cells, unlike the tube with freshly prepared saponin (A). Storage at -20°C has no negative impact on the ability to lyse human cells and allows the lysing property of the saponin to be preserved over time.

[0098] Example 5 - Removal of polypeptides by heat treatment and a high amount of heat-resistant protease.

[0099] The purpose of this test is to demonstrate the improved efficiency of the method according to the present invention when it includes the addition of a heat-resistant protease coupled with a heat treatment.

[0100] The test protocol is as follows. A 9 mL sample of whole blood is treated by the previously described double lysis method. 9 mL samples of whole blood are lysed with 8 mL of saponin solution and then centrifuged for 20 minutes at 24,000 g. After removal of the supernatant, the pellets are treated with 400 pL of saponin solution and then with a nuclease. The polypeptides present in the sample are then removed by treatment for 10 minutes at 80°C with an unusually high amount of papain (60,000 units), a heat-resistant protease. Samples of 0.2 pL of treated blood are taken at different stages of treatment, after the first lysis with a saponin solution (panel A), after the second lysis with a saponin solution (panel B), and after the 10-minute treatment at 80°C with 60,000 units of papain (panel C) and are analyzed by SDS-PAGE to evaluate their polypeptide content.The second lysis step with a saponin solution partially reduces the amount of polypeptides present in the sample.

[0101] The results are presented in [Fig. 5]. Treatment with a large quantity of papain at high temperature eliminates all the polypeptides present in the sample in just 10 minutes (panel C).

Claims

Demands

1. Method for purifying DNA from microorganisms of interest present in a biological sample, said method comprises the following steps: a. bringing a biological sample into contact with a saponin solution, to lyse the animal cells included in said biological sample, b. centrifuging the mixture from step a), removing the supernatant and retaining the pellet containing the cells of microorganisms of interest, c. bringing the pellet into contact with a saponin solution in order to lyse the animal cells not lysed in step a), d. removing the animal DNA by nuclease treatment, e. inactivating the nuclease.

2. Method according to the preceding claim, characterized in that the volume of biological sample is between 0.1 mL and 40 mL.

3. A method according to any one of the preceding claims, characterized in that the biological sample is a fresh biological sample selected from blood, cerebrospinal fluid, bronchoalveolar lavage fluid, urine, stool, biopsies and mixtures thereof.

4. Method according to any one of the preceding claims, characterized in that the biological sample is a whole blood sample.

5. Method according to any one of the preceding claims, characterized in that the amount of nuclease is between 100A3 and 210A6 units (U) per biological sample.

6. Method according to any one of the preceding claims, characterized in that the method further comprises a step of lysis of the cells of microorganisms present, after the steps of removal of animal DNA and inactivation of nuclease.

7. Method according to the preceding claim, characterized in that the lysis is carried out by means of beads.

8. Method according to the preceding claim, characterized in that the diameter of the balls is between 1 mm and 6 mm.

9. Method according to any one of the preceding claims, characterized in that the saponin solution is a saponin solution in fresh or frozen form.

10. A method according to any one of the preceding claims, characterized in that the method comprises an additional step of removing polypeptides present in the biological sample, after the step of removing animal DNA, said polypeptide removal step comprising the addition of a heat-resistant protease followed by heat treatment.

11. Method according to the preceding claim, characterized in that the heat-resistant protease is papain.

12. Method according to any one of claims 10 or 11, characterized in that the temperature of the heat treatment is between 10°C and 100°C.

13. Method according to any one of claims 10 to 12, characterized in that the polypeptide removal step further comprises the addition of at least one reducing agent selected from DTT, DTE, Beta-mercaptoethanol, TCEP, glutathione, and Thioglycerol.

14. Method according to any one of the preceding claims, characterized in that the method comprises a final step of purifying DNA from microorganisms by means of a heat treatment.

15. Method according to the preceding claim, characterized in that the temperature of the heat treatment is between 60°C and 100°C, preferably 90°C.

16. Method according to any one of the preceding claims, characterized in that steps a) and b) are repeated at least once before treatment with nucleases.

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