Specific isolation method of nucleic acids of interest

Description

[0001] The present invention relates to a method for selectively isolating microorganisms of interest and / or nucleic acids of interest from a liquid biological sample containing or likely to contain, in particular, a large number of non-targeted cells and / or a large number of nucleic acids of non-targeted cells. It also relates to their use, diagnostic tests based on a method for isolating microorganisms of interest or on a method for isolating nucleic acids of microorganisms of interest and kits for enabling the isolation of microorganisms of interest and / or nucleic acids of microorganisms of interest from a liquid biological sample comprising or likely to comprise, in particular, microorganisms of interest, non-targeted cells and, optionally, debris of microorganisms of interest and / or non-targeted cells.

[0002] The state of the art consists of a number of scientific publications and patents. For example, in 1992, Baker and colleagues described the use of a reagent called saponin to differentially lyse human cells and isolate the Plasmodium pathogen from whole blood. They demonstrated that saponin at a low final concentration, around 0.015%, could lyse human cells in 20 µL of citrate-buffered whole blood and provide enough clean lysate for PCR detection.

[0003] Patent application EP-A-0.745.849 revisited this differential approach of using saponin for larger whole blood volumes (5 mL) with a final concentrated saponin solution in the order of 0.020-0.125%.

[0004] Another patent application EP-A-2,185,681 proposes a method for preparing saponin solutions, using a more concentrated saponin solution, i.e. with a final concentration of between 2 and 10% for an average-sized volume of whole blood, i.e. 5 mL. This invention proposes to use hypotonic or physiological buffers for such saponin solutions.

[0005] However, today, no rapid solution is available to isolate and identify very small quantities of pathogenic cells from large biological samples. For example, for sepsis, the need is to be able to identify 1 to 10 colony-forming units (CFU) of pathogens in 10 mL of whole blood within 6 hours, or in the most difficult cases 1 fg of targeted pathogen nucleic acid for more than 800 µg of non-targeted (human) nucleic acids.

[0006] The problems encountered in the diagnosis of infectious diseases are therefore: 1) the complexity of the matrix sample usually including very few pathogen cells on the one hand, and a very large number of human cells, and other cells or particles on the other hand, which must be removed so as not to interfere with the isolation and identification of pathogens, and 2) the physical limitations of isolating a very small number of pathogens from a very large sample volume. In summary, the challenge is to eliminate as many non-targeted elements (cells and / or particles) as possible while keeping the maximum number of pathogenic targets that were initially low in number in order to artificially increase their concentration and facilitate their detection.

[0007] The invention described herein is a method that can isolate and identify a small number of pathogens from large volumes of biological samples (e.g., of the order of 10 ml) in a relatively short time. These pathogens include, but are not limited to, bacteria, viruses, and fungi. The invention can identify as low as 3 CFU in 10 ml of whole blood (i.e., 0.3 CFU / ml), with a detection limit never before described in the literature. Its novelty is based on: 1. An attractive detection limit never before achieved in the order of 0.3 CFU / mL, in relation to the clinical need which is 0.1 to 1 CFU / mL. 2. Ability to use different whole bloods buffered with EDTA or Citrate but also with heparin. Heparin is a reagent widely used in hospitals, but often discarded in molecular biology approaches due to its ability to prevent nucleic acid amplification and enzyme-based reactions. This invention can handle heparin-buffered blood, which is not generally the case for molecular methods described in the literature. 3. Ability to use large volumes of whole blood of 10 mL or more. 4. The use of polyethylene glycol (PEG) to enhance pathogen precipitation. Precipitation of DNA or viruses by PEG is very well described in the literature.However, the use of PEG to precipitate bacterial cells or fungi such as yeasts is absent in the state of the art. Since saponin is a detergent-like reagent, pathogenic cells or particles may float inside the tube or have difficulty precipitating to the bottom of a tube under centrifugation. Even if some treated samples do not require it, the addition of PEG ensures a precipitation of targets that is constant without the risk of flotation. In addition, this addition of PEG improves the adhesion of pellets containing pathogens to the plastic tube and the resistance to surface washing. 5. Combine differential lysis with saponin with highly concentrated nuclease treatments; in other words, lyse non-target cells that will release their nucleic acids, which in a second step will be degraded by nuclease treatment.This differential lysis releases a large amount of human nucleic acids. Typically 880 µg of human DNA can be extracted from 10 ml of whole blood. These human nucleic acids are the source of significant problems when the aim is to detect a very small number of copies of pathogenic nucleic acids: i) they compete during the purification of pathogenic target nucleic acids by saturating the nucleic acid capture reagents due to their excess (such as silica membranes, magnetic silica beads, chromatography or affinity matrices, etc.), ii) they interfere during the amplifications of the pathogenic molecules. By eliminating the maximum amount of human nucleic acids, the detection limits are improved. According to these critical points, the invention uses a large amount of nucleases to eliminate a maximum of human nucleic acids after lysis by saponin.The combination of saponin with highly concentrated nuclease treatment has never been described in the literature. 6. Finally, we have demonstrated that buffered saponin solution, with a pH between 6 and 9, prevents the precipitation of unwanted blood elements, particularly red blood cells. Scientists have previously used buffers close to pH 7 for saponin solution, but without ever correlating the impact of this pH with sample stability.

[0008] In summary, the present invention combines different uses of chemicals to achieve a pathogen detection limit compatible with detection values ​​in a clinical range (0.1 to 1 CFU / mL). The combined use of saponin with PEG and at least one nuclease has never been described and allows a technical improvement unknown to date. The combination of these reagents can be complete (saponin + PEG + nuclease) or partial (saponin + nuclease or saponin + PEG only or PEG alone) to prepare the sample for identification analysis or detection.

[0009] For this purpose, the present disclosure relates to a method for selectively isolating microorganisms of interest from a liquid biological sample comprising or likely to comprise, in particular: microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and non-targeted elements, i.e.: non-targeted cells whose cell membrane contains cholesterol, and possibly, viruses with envelopes containing cholesterol, and possibly mycoplasmas containing cholesterol, and possibly, debris of microorganisms of interest and / or non-targeted cells, the method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol or the viral envelopes containing cholesterol or the mycoplasma membranes containing cholesterol, b) carrying out an osmotic shock of the non-targeted cells in order to lyse them specifically, c) adding a solution of at least one enzyme capable of lysing the free nucleic acids (DNA and / or RNA) from the non-targeted elements lysed in solution in the sample, making it possible to selectively obtain the microorganisms of interest. The present disclosure also covers a method for selectively isolating microorganisms of interest from a liquid biological sample comprising or likely to comprise, in particular: microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and non-targeted elements, i.e.: non-targeted cells whose cell membrane contains cholesterol, and possibly, viruses with envelopes containing cholesterol, and possibly mycoplasmas containing cholesterol, and possibly, debris of microorganisms of interest and / or non-targeted cells, the method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol and / or the viral envelopes containing cholesterol and / or the membranes of mycoplasmas containing cholesterol, b) carrying out an osmotic shock of the non-targeted cells in order to lyse them specifically, c) adding an agent for precipitating the non-lysed microorganisms of interest in solution in the sample, making it possible to selectively obtain the microorganisms of interest. The invention covers a method for selectively isolating microorganisms of interest from a liquid biological sample comprising or likely to comprise, in particular: microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and non-targeted elements, i.e.: non-targeted cells whose cell membrane contains cholesterol, and possibly, enveloped viruses containing cholesterol, and possibly mycoplasmas containing cholesterol, and possibly, debris of microorganisms of interest and / or non-targeted cells, the method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol and / or the viral envelopes containing cholesterol and / or the membranes of mycoplasmas containing cholesterol, b) carrying out an osmotic shock of the non-targeted cells in order to lyse them specifically, c) adding an agent for precipitating the non-lysed microorganisms of interest in solution in the sample, which is polyethylene glycol and d) adding a solution of at least one enzyme capable of lysing the free nucleic acids (DNA and / or RNA) from the non-targeted elements lysed in solution in the sample, making it possible to selectively obtain the microorganisms of interest. Step c) of the latter method can be carried out independently of step a), after steps a) and b) or after step d).

[0010] The present disclosure also relates to a method for selectively isolating nucleic acids of interest from a liquid biological sample comprising or likely to comprise, in particular: microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and non-targeted elements, i.e.: non-targeted cells whose cell membrane contains cholesterol, and possibly, viruses with envelopes containing cholesterol, and possibly mycoplasmas containing cholesterol, and possibly, debris of microorganisms of interest and / or non-targeted cells, the method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol and / or viral envelopes containing cholesterol and / or the membranes of mycoplasmas, b) carrying out an osmotic shock of the non-targeted cells in order to lyse them specifically, c) adding a solution of at least one enzyme capable of lysing the free nucleic acids (DNA and / or RNA) derived from the non-targeted elements lysed in solution in the sample, d) inactivating the enzyme added in step (c), and e) making accessible the nucleic acids of microorganisms of interest not degraded by the enzyme of step (c).

[0011] According to a fifth embodiment, the disclosure covers a method for selectively isolating nucleic acids of interest from a liquid biological sample comprising or likely to comprise, in particular: microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and non-targeted elements, i.e.: non-targeted cells whose cell membrane contains cholesterol, and possibly, enveloped viruses containing cholesterol, and possibly mycoplasmas containing cholesterol, and possibly, debris of microorganisms of interest and / or non-targeted cells, the method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol and / or the viral envelopes containing cholesterol and / or the membranes of mycoplasmas containing cholesterol, b) carrying out an osmotic shock of the non-targeted cells in order to lyse them specifically, c) adding an agent for precipitating the non-lysed microorganisms of interest in solution in the sample, and d) making accessible the nucleic acids of microorganisms of interest not accessible by the action of steps a) and b).

[0012] The invention also covers a method for selectively isolating nucleic acids of interest from a liquid biological sample comprising or likely to comprise, in particular: microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and non-targeted elements, i.e.: non-targeted cells whose cell membrane contains cholesterol, and possibly, enveloped viruses containing cholesterol, and possibly mycoplasmas containing cholesterol, and possibly, debris of microorganisms of interest and / or non-targeted cells, the method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol and / or the viral envelopes containing cholesterol and / or the membranes of mycoplasmas containing cholesterol, b) carrying out an osmotic shock of the non-targeted cells in order to lyse them specifically, c) adding an agent for precipitating the non-lysed microorganisms of interest in solution in the sample which is polyethylene glycol, and d) adding a solution of at least one enzyme capable of lysing the free nucleic acids (DNA and / or RNA) from the non-targeted elements lysed in solution in the sample, e) inactivating the enzyme added in step (d), f) obtaining the nucleic acids of microorganisms of interest: not degraded by the enzyme of step (d), and not accessible by the action of steps a) and b). Step c) of the latter method can be carried out independently of step a), after steps a) and b) or after step d).

[0013] According to a seventh aspect, the present disclosure also relates to an improved method for precipitating microorganisms of interest chosen from bacteria and fungi (preferably yeasts), consisting of adding at least one precipitation agent to liquid biological samples, where appropriate previously treated with saponin.

[0014] The addition of precipitation agent(s) improves the precipitation yield of microorganisms contained in a liquid biological sample.

[0015] Regardless of the embodiment, the isolation method is characterized in that the saponin is in a volume at least equal to or greater than the volume of the sample at equal concentration.

[0016] The final saponin concentration is greater than 0.02% and less than or equal to 20%, preferably between 0.05% and 20%, more preferably between 0.5 and 20% and even more preferably between 0.08 and 4%.

[0017] Osmotic shock of non-targeted cells to specifically lyse them is an inherent property of contacting the liquid biological sample with the saponin formulation as described in step a). This saponin formulation is used in large volumes which has the effect of: to weaken or destabilize cell membranes containing cholesterol, namely the membranes of non-targeted cells, and at the same time to induce osmotic pressure which will lead to the turgor of non-targeted cells and their lysis. In other words, steps a) and b) can be rewritten into a single step consisting of contacting the liquid biological sample with a saponin formulation in order to specifically lyse the cholesterol-containing cell membranes of non-target cells and the cholesterol-containing viral envelopes and the cholesterol-containing membranes of mycoplasmas.

[0018] The isolation process is also characterized in that the saponin consists of a triterpenoid. Saponin is specific to membranes or envelopes containing cholesterol.

[0019] In the method for isolating microorganisms of interest and the improved method for precipitating microorganisms of interest according to the disclosure, the precipitation agent used is selected from PEG, glycogen and nucleic acids (DNA / tRNA, etc.). It is also possible to use them as a mixture.

[0020] The precipitating agent used in the context of the invention is PEG.

[0021] In the process of isolating nucleic acids from microorganisms of interest, the precipitation agent may be polyethylene glycol (PEG).

[0022] The concentrations of precipitation agent used in the context of the invention are between 0.1% and 20%, preferably between 1% and 20%.

[0023] At the end of the process of isolating the microorganisms of interest, whatever its implementation, it is possible to treat the medium obtained to further isolate the microorganisms of interest, thus allowing their detection. This is done using conventional cellular techniques, for example cytology techniques (flow cytometry or others), immunology techniques (technology with antibodies or phages for detection by immunoassay) or conventional cell culture techniques. Thus, it is possible to apply the medium obtained for conventional culture on an appropriate medium, preferably on a solid medium (for example a nutrient agar medium) in a Petri dish for at least 24 hours, for example 48 hours for yeasts, at a suitable temperature, for example 30°C for yeasts, preferably 37°C, or any medium suitable for the growth of the microorganisms of interest.Any known technique and protocol allowing the growth of microorganisms and their individualization / isolation can also be used.

[0024] According to certain variants of the isolation process, the enzyme capable of lysing free nucleic acids is an enzyme which is then inactivated: chemically by the addition of EDTA and / or EGTA and / or DTT and / or β-mercaptoethanol and / or DEPC, and / or guanidine, and / or physically by increasing the temperature between 40 and 100°C in the presence or absence of detergents, such as Sodium Docecyl Sulfate (SDS).

[0025] In cases where the isolation method according to the invention comprises only one enzyme capable of lysing the free nucleic acids (DNA and / or RNA) derived from the non-targeted elements lysed in solution in the sample, this enzyme is a DNAse.

[0026] According to certain variant embodiments of the isolation method where at least one enzyme capable of lysing the free nucleic acids is added, i.e. during step (c) when the method used does not include a step of adding a precipitation agent or during step (d) when the method includes a step of adding a precipitation agent, the enzyme capable of lysing the free nucleic acids is an enzyme which can be inactivated: chemically by the addition of EDTA and / or EGTA and / or DTT and / or β-mercaptoethanol and / or DEPC, and / or guanidine, and / or physically by increasing the temperature between 40 and 100°C in the presence or absence of detergents, such as Sodium Docecyl Sulfate (SDS).

[0027] In the methods for isolating microorganisms of interest and for isolating nucleic acids from microorganisms of interest comprising a step of adding a precipitation agent and a step of adding at least one enzyme capable of lysing the free nucleic acids (DNA and / or RNA) from the non-targeted elements lysed in solution in the sample, these two steps do not have a defined order and can be interchanged in the course of the method. In the context of the method for isolating nucleic acids from microorganisms of interest comprising a step of inactivating the enzyme capable of lysing the free nucleic acids, the precipitation agent can be added after this inactivation step.

[0028] The invention also relates to a method for the isolation of microorganisms of interest or for the isolation of nucleic acids of microorganisms of interest or for the precipitation of microorganisms of interest, in a liquid biological sample, preferably blood, characterized in that during the method, the pH is maintained in a range between 5 and 10, preferably between 6 and 9, by the addition of a solution: basic if the pH value is less than 5, preferably less than 6, acidic if the pH value is greater than 10, preferably greater than 9, so that the pH is within the range.

[0029] According to a variant of use, the disclosure uses a saponin formulation leading to a final concentration greater than 0.02% and less than or equal to 20%, preferably greater than 0.05% and less than or equal to 20% and / or a precipitating agent (or precipitation agent) at a concentration of 0.1 to 20%, preferably 0.1 to 4%, more preferably still 0.5 to 4%, and / or an enzyme capable of lysing free nucleic acids (DNA and / or RNA) containing between 500 and 20,000 enzymatic units.

[0030] According to another of its aspects, the invention covers the use of a formulation of saponin and a solution of at least one enzyme capable of lysing free nucleic acids (DNA and / or RNA) and at least one precipitation agent which is PEG, for the isolation of microorganisms of interest or nucleic acids of microorganisms of interest, in a liquid biological sample.

[0031] According to another of its aspects, the present invention covers the use of PEG for the precipitation of microorganisms of interest, preferably bacteria and fungi (preferably yeasts), in liquid biological samples, where appropriate previously treated with saponin.

[0032] The invention also relates to a diagnostic test based on a method for isolating microorganisms of interest, as described above, or on the uses for isolating microorganisms of interest, as described above, or on a method for precipitating microorganisms as described above.

[0033] The invention also relates to a diagnostic test based on a method for isolating nucleic acids from microorganisms of interest, as described above, or to the uses for isolating nucleic acids from microorganisms of interest, as described above.

[0034] The diagnosis can be carried out using conventional techniques known to those skilled in the art, for example, implementing conventional detection techniques used in microbiology, immunoassay techniques or conventional molecular biology techniques such as PCR, NASBA, etc.

[0035] The invention also relates to a diagnostic kit for enabling the isolation of microorganisms of interest within a liquid biological sample comprising or likely to comprise, in particular, microorganisms of interest, non-targeted cells and, optionally, enveloped viruses, mycoplasmas and / or debris of microorganisms of interest and / or non-targeted cells, the kit comprising: (a) a container, and (b) at least one saponin formulation, and (c) at least one solution of a precipitating agent, which is polyethylene glycol (PEG) (d) at least one enzyme capable of lysing nucleic acids, and (e) the kit for detecting the microorganisms of interest and / or the nucleic acids of microorganisms of interest.

[0036] The invention finally relates to a diagnostic kit for enabling the isolation of nucleic acids of microorganisms of interest within a liquid biological sample comprising or likely to comprise, in particular, microorganisms of interest, non-targeted cells and, optionally, enveloped viruses, mycoplasmas and / or debris of microorganisms of interest and / or non-targeted cells, the kit comprising: (a) a container, and (b) at least one saponin formulation, and (c) at least one solution of a precipitating agent, which is polyethylene glycol (PEG), (d) at least one enzyme capable of lysing nucleic acids, and (e) the kit for detecting the microorganisms of interest and / or the nucleic acids of microorganisms of interest.

[0037] The two kits described above may further comprise (d) at least one solution of at least one enzyme capable of lysing nucleic acids.

[0038] The kit also includes: (c) or (d') at least one acidic solution and / or at least one basic solution, and / or (d) EDTA and / or EGTA and / or DTT and / or β-mercaptoethanol and / or DEPC, and / or guanidine and / or (e) at least one detergent or anionic agent.

[0039] In the present description, the enzyme(s) for lysing the nucleic acids are enzymes known and conventionally used by those skilled in the art, such as DNAse I, RNAse If, ​​etc., and the step of making the nucleic acids of the microorganisms of interest accessible is carried out in a conventional manner known to those skilled in the art, for example by mechanical lysis followed by purification of the nucleic acids and PCR amplification or any other known technique.

[0040] In the remainder of this patent application, the terms used are given the following definitions: By "viral envelope" we mean the envelope of enveloped viruses, which contains cholesterol, unlike the capsid of capsid viruses, which does not. The viral envelope is therefore sensitive to saponin. By "destabilizing cell membranes and / or viral envelopes and / or mycoplasma membranes" we mean the physicochemical mechanisms leading to a loss of osmolarity regulation at the membrane level and / or the viral envelope and / or the membrane of a mycoplasma, a disruption of membrane transport and the potential appearance of pores at the level of the cell membrane, mycoplasma or viral envelope. The term "microorganisms of interest" includes all microorganisms that do not contain accessible cholesterol, which are potentially pathogenic, particularly for humans.These microorganisms include viruses (except enveloped viruses), bacteria, fungi (yeasts) and also microscopic animals. The "nucleic acids of interest" correspond to the nucleic acids (DNA and RNA) contained in the cells or particles of the "microorganisms of interest" defined above. By "non-targeted cells" we mean all cells of living organisms that do not contain "nucleic acids of interest" as defined above. The abbreviation "EDTA" corresponds to ethylenediamine tetraacetic acid (corresponding in English to . to EthyleneDiamineTetraacetic Acid ). The abbreviation "EGTA" stands for ethylene glycol tetraacetic acid (in English Ethylene Glycol Tetraacetic Acid). The abbreviation "DTT" stands for Dithiothreitol. The abbreviation "DEPC" stands for Diethylpyrocarbonate. The abbreviation CFU stands for Colony-Forming Unit. The term "detergents" refers to all classes of molecules that can induce a physicochemical modification of other molecules. These detergents can be chemical in nature, such as SDS, Tween-20, Tritonx-100, Brij97, or enzymatic. By "liquid biological sample" is meant a liquid sample likely to contain the "microorganisms of interest" selected from the following group: amniotic fluid, aqueous humor, bile, blood, mammary secretion, bronchoalveolar lavage, cerebrospinal fluid, chyle, chyme, feces, interstitial fluid, lymph, menstruation, mucus, plasma, pleural fluid, pus, saliva, sebum, semen, serum, sputum, sweat, synovial fluid, tear, urine and vitreous humor. Furthermore, it should be clear that the method for selective isolation of nucleic acids of interest as described above and its use allow the isolation of microorganisms of interest, to the extent that the nucleic acids of interest correspond to the nucleic acids (DNA and / or RNA) contained in the cells or particles of the microorganisms of interest. In other words, the selective isolation of nucleic acids of interest allows the isolation of the microorganisms of interest from which the nucleic acids of interest originate.

[0041] The attached examples are presented to demonstrate the effectiveness of the method according to the invention and are given for illustrative purposes and not as a limitation. Example 1 : Detection of 5 and 10 CFU of Pseudomonas aeruginosa in 10 mL of whole blood treated with 4% saponin solution

[0042] Inside a 50 mL plastic tube, 20 mL of EDTA-treated whole blood was inoculated with 20, 10, 2, and 0 (negative control) CFU of Pseudomonas aeruginosa.The number of CFU inserted into the blood was verified by plating on agar media in Petri dishes. The 20 mL of inoculated blood was divided in half and each 10 mL volume was deposited into two 50 mL plastic tubes to have duplicates. Forty milliliters of a filtered solution of 4% saponin, 50 mM Tris-HCl at pH 8.0 and 4% PEG-8000 was added to the inoculated blood. The tubes were shaken by inversion twice, incubated at room temperature for 5 minutes and centrifuged at 12000 g for 10 minutes. The supernatant was discarded and the sticky pellet was washed three times with 15 mL of 4% PEG-8000 which prevents the pellet from detaching. A volume of 200 µL of 10 mM Tris-HCl at pH 7.5 was added to the pellet.

[0043] Subsequently, 10 µL of DNAse I (500 u / µL; Roche) and 2 µL RNAse If (50 u / µL; New England Biolabs) were added to the tubes. The pellets were digested with the enzymes for 10 minutes with two vortexings after 2 minutes and 4 minutes of incubation. After nuclease digestion, 10 µL of 0.5 M EDTA, pH 8.0, was added to the tubes. The samples were transferred to 1.5 mL microcentrifuge tubes containing 200 mg of 1 mm diameter glass beads and 50 mg of 0.1 mm diameter zirconium beads. The microcentrifuge tubes were heated for 10 minutes at 80°C. 20 µL of Proteinase K (Novagen) and 40 µL of 10% SDS were then added to the tubes. The microtubes were incubated for 5 minutes at room temperature and heated for 5 minutes at 80°C.

[0044] Mechanical lysis of cells of Pseudomonaswas performed by shaking the tubes containing the beads for 20 minutes using a vortex. The DNA present in the lysis supernatant was purified using the Nucleospin Blood ® kit from Macherey-Nagel. Quantitative PCR amplification was performed using the entire eluate, i.e. 40 µl. Samples at 5 CFU of Pseudomonas aeruginosa were indeed detected on a replica using the invention, and a partial detection of one replica out of two was observed for 1 CFU inserted, as is clearly presented in the following Table I: Tab water I: Evaluation of the detection limit for Pseudomonas aeruginosa at using the method according to the invention Sample Cq Number of replicas detected 10 CFU total 38,78 2 / 2 10 CFU total 37, 74 5 CFU total 42,18 2 / 2 5 CFU total 37,84 1 CFU total 41,73 1 / 2 1 CFU total N / A* Negative control N / A* 0 / 2 Negative control N / A* *: no fluorescence signal was detected after 50 amplification cycles. Example 2 : Detection of 28 and 140 CFU of Candida albicans in 10 mL of whole blood treated with 4% saponin solution

[0045] Inside a 50 mL plastic tube, 20 mL of EDTA-treated whole blood was inoculated with 140, 28, and 0 (negative control) CFU of Candida albicans.The number of CFU inserted into the blood was verified by plating on agar media in Petri dishes. The 20 mL of inoculated blood was divided in half and each 10 mL volume was deposited into two 50 mL plastic tubes to obtain duplicates. Forty milliliters of a filtered solution of 4% saponin, 50 mM Tris-HCl at pH 8.0 and 4% PEG-8000 were added to the inoculated blood. The tubes were shaken by inversion twice, incubated at room temperature for 5 minutes and centrifuged at 12000 g for 10 minutes. The supernatant was discarded and the pellet was washed three times with 15 mL of 4% PEG-8000 which prevents the pellet from detaching. A volume of 200 µL of 10 mM Tris-HCl at pH 7.5 was added to the pellet. Subsequently, 10 µL of DNAse I (500 µ / µL; Roche) and 2 µL RNAse If (50 µ / µL; New England Biolabs) were added to the tubes.Pellets were digested with enzymes for 10 minutes with two vortexings after 2 minutes and 4 minutes of incubation. After nuclease digestion, 10 µL of 0.5 M EDTA pH 8.0, 40 µL of 10% SDS and 20 µL of Proteinase K were added to the tubes. Samples were transferred to 1.5 mL microcentrifuge tubes containing 200 mg of 1 mm diameter glass beads and 50 mg of 0.1 mm diameter zirconium beads. The microcentrifuge tubes were heated for 60 minutes at 80°C. Mechanical lysis of cells. Candida albicans was performed by shaking the beads for 20 minutes using a vortex. The DNA present in the lysis supernatant was purified using the Macherey-Nagel Nucleospin Blood ®< kit and purified a second time using the Macherey-Nagel gDNA Clean-up XS ®< kit. Quantitative PCR amplification was performed using the entire eluate, i.e. 40 µL. The 28 CFU samples of Candida albicanswere detected on a replica using the invention, as is well represented in the following Table 2: Table 2 : Detection for Candida albicans using the method according to the invention Sample Cq Number of replicas detected 140 CFU total 37, 44 2 / 2 140 CFU total 36,78 28 CFU total 40,08 2 / 2 28 CFU total 38,42 Negative control N / A* 0 / 1 *: no fluorescence signal was detected after 50 amplification cycles. Example 3 : Detection of 20,000 human Adenovirus 5 virions in 10 mL of whole blood treated with a 4% saponin solution

[0046] Inside a 50 mL plastic tube, 10 mL of EDTA-treated whole blood was inoculated with 20,000 human Adenovirus 5 virons. Forty milliliters of a filtered 4% saponin solution with 50 mM Tris-HCl at pH 8.0 and 4% PEG-8000 were added to the inoculated blood. The tubes were inversion-mixed twice, incubated at room temperature for 5 minutes, and centrifuged at 12,000 g for 10 minutes. The supernatant was discarded, and the pellet was washed three times with 15 mL of 4% PEG-8000, which prevents pellet detachment. A volume of 200 µL of 10 mM Tris-HCl at pH 7.5 was added to the pellet. Subsequently, 10 µL of DNAse I (500 µ / µL; Roche) and 2 µL RNAse If (50 µ / µL; New England Biolabs) were added to the tubes. The pellets were digested with the enzymes for 10 minutes with two vortexings after 2 minutes and 4 minutes of incubation.After nuclease digestion, 10 µL of 0.5 M EDTA, pH 8.0, and 40 µL of 10% SDS were added to the tubes. Samples were transferred to 1.5 mL microtubes containing 200 mg of 1 mm diameter glass beads and 50 mg of 0.1 mm diameter zirconium beads. The microtubes were heated for 10 minutes at 80°C. Mechanical lysis of virions was performed by shaking the tubes containing the beads for 20 minutes using a vortex mixer. DNA in the lysis supernatant was purified using the Macherey-Nagel Nucleospin Blood ® kit. Nucleic acids were eluted with 40 µL. Quantitative PCR amplification was performed using 10 µl of eluate. The 20,000 virions were indeed detected using the invention, see Table 3 below: . Table 3 Evaluation of the detection limit of human Adenovirus 5 using the invention : Sample Cq Number of replicas detected 20,000 virions total 34,7 1 / 1 Example 4 : Lysis efficiency of human blood cells by different concentrations of saponin.

[0047] Inside a 50 mL plastic tube, 10 mL of EDTA-treated whole blood was inoculated with 24 and 0 (negative control) CFU of Pseudomonas aeruginosa.The number of CFU inserted into the blood was verified by plating on agar media in Petri dishes. Forty milliliters of a filtered solution of saponin, 50 mM Tris-HCl at pH 8.0 and 4% PEG-8000 were added to the inoculated blood. The final saponin concentrations were 0.005%, 0.02%, 0.08% and 0.4%. Each concentration was tested in duplicate. The tubes were inversion-mixed three times, incubated at room temperature for 10 minutes and centrifuged at 12000 g for 10 minutes. The supernatant was discarded and the sticky pellet was washed three times with 15 mL of 4% PEG-8000 which prevents the pellet from detaching. A volume of 400 µL of 10 mM Tris-HCl at pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 5000 u DNAse I (Roche), 100 u RNAse If (New England Biolabs) was added to the pellet. The pellets were incubated at 32°C with shaking for 90 minutes.Samples were transferred into microtubes containing 10 µL of 0.5 M EDTA pH 8.0 and 400 µL of buffer B3 (Macherey-Nagel). The microtubes were heated for 10 minutes at 80°C and then cooled for 5 minutes on ice. 5 µg of lysine was added to the tubes. The microtubes were incubated for an additional 10 minutes on ice and then treated with the Nucleospin Blood ® kit from Macherey-Nagel according to the supplier's conditions, however, doubling the quantities of proteinase K and ethanol to respect the reagent ratios. Quantitative PCR amplification was performed using 2 µL of eluate for the detection of human DNA and 38 µL for PCR detecting DNA from . Pseudomonas aeruginosa.

[0048] The majority of tubes were positive for Pseudomonas aeruginosa (the negative controls, non-inoculated blood, treated with 0.4% saponin being negative). The example shows that it is possible to detect Pseudomonas aeruginosafor saponin concentrations well below 0.4%.

[0049] Amplifications for final saponin concentrations equal to or greater than 0.08% failed to detect human DNA or extremely low amounts (Table 4). On the contrary, for final saponin concentrations equal to or less than 0.02%, the amounts of human DNA detected are very high (Cq around 28). This demonstrates that at a final saponin concentration less than or equal to a value of 0.02%, white blood cells are no longer properly lysed whereas they are very well lysed with a final concentration of 0.08% or 0.4% saponin. Tab water 4: Evaluation of human blood cell lysis Sample Cq (human DNA) Number of positive tubes for P . aeruginosa Negative control 0.4% saponin Not detected* 0 / 2 (Controls) Negative control 0.4% saponin Not detected* 0.4% saponin 39, 16 2 / 2 0.4% saponin Not detected* 0.08% saponin Not detected* 1 / 2 0.08% saponin Not detected* 0.02% saponin 27,35 2 / 2 0.02% saponin 27,99 0.005% saponin 28,63 1 / 2 0.005% saponin 29,27 *: no fluorescence signal was detected after 50 amplification cycles. Example 5 : Detection of 21 CFU of Pseudomonas aeruginosa in 10 mL of whole blood treated with a 0.4% saponin solution.

[0050] Inside a 50 mL plastic tube, 10 mL of EDTA-treated whole blood was inoculated with 21 (5 tubes) and 0 (negative control, 1 tube) CFU of Pseudomonas aeruginosa.The number of CFU inserted into the blood was verified by plating on agar media in Petri dishes. Forty milliliters of a filtered solution of 0.4% saponin, 50 mM Tris-HCl at pH 8.0 and 4% PEG-8000 were added to the inoculated blood. The tubes were shaken by inversion three times, incubated at room temperature for 10 minutes and centrifuged at 12000 g for 10 minutes. The supernatant was discarded and the sticky pellet was washed three times with 15 mL of 4% PEG-8000 which prevents the pellet from detaching. A volume of 800 µL of 10 mM Tris-HCl at pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 5000u DNAse I (Roche), 100u RNAse If (New England Biolabs) was added to the pellet. The pellets were incubated at 32°C with shaking for 90 minutes. 10 µL of 0.5 M EDTA pH 8.0 and 400 µL of buffer B3 (Macherey-Nagel) were added and the tubes were heated for 10 minutes at 80°C and then cooled for 5 min in ice. 5 µg of lysine was added to the tubes.The tubes were incubated for an additional 10 minutes on ice and then treated with the Macherey-Nagel Nucleospin Blood ® kit according to the supplier's conditions, however quadrupling the quantities of proteinase K and ethanol to respect the reagent ratios. Quantitative PCR amplification was carried out using 2 µl of eluate for the detection of human DNA and 38 µL for PCR detecting DNA. Pseudomonas aeruginosa. Tab water 5 : Quantitative PCR detection of P DNA. aeruginosa Sample Cq (ADN P. aeru. ) Number of positive tubes for P . aeruginosa Negative control Not detected* 0 / 1 21 CFU total 36,60 5 / 5 21 CFU total 36,17 21 CFU total 37,21 21 CFU total 38,81 21 CFU total 38,36 *: no fluorescence signal was detected after 50 amplification cycles. Example 6: Detection by growth on solid medium of 117 CFU of Candida albicans from 10 mL of whole blood treated with 4% saponin

[0051] Inside a 50 mL plastic tube, 10 mL of EDTA-treated whole blood was inoculated with 117 CFU of Candida albicans.The number of CFU inserted into the blood was verified by plating on agar media in Petri dishes. Forty milliliters of a filtered solution of 4% saponin, 50 mM Tris-HCl at pH 8.0, and 4% PEG-8000 were added to the inoculated blood. The tubes were shaken by inversion twice, incubated at room temperature for 5 minutes, and centrifuged at 12,000 g for 10 minutes. The pellets obtained were resuspended with 212 µl of Tryptone salt (AES) or 212 µl of nuclease mix (Tris 10 mM pH7.5; DNAse I (Roche) 5000 u; RNAse If (New England Biolabs) 100 u), incubated for 10 minutes at room temperature and then spread on a solid medium in SDC petri dishes (bioMérieux). The number of colonies was then counted after incubation at 30°C for 48 hours. Table 6 shows that on average 66% of CFU were found after saponin treatment with or without nuclease treatment. Table 6 Enumeration on box of colonies of Candida albicans after treatment with 4% saponin : Sample Resuspension Number of colonies Average 117 CFU total Tryptone salt 84 78.5 (67% of 117) 117 CFU total Tryptone salt 73 117 CFU total Nuclease mix 87 76 (65% of 117) 117 CFU total Nuclease mix 65 *: no fluorescence signal was detected after 50 amplification cycles. Example 7: Facilitating effect of PEG on the precipitation by centrifugation of Pseudomonas aeruginosa present in blood

[0052] 200µL of EDTA-treated whole blood was dispensed into 1.5 mL plastic tubes. 1 mL of 10 mM MgCl2 or 1 mL of MgCl2 supplemented with 4% final PEG was added to the tubes. Subsequently, these tubes were inoculated with 129 CFU of Pseudomonas aeruginosa. The tubes were vortexed for 5 seconds and then centrifuged for 10 minutes at 5000g. The pellets were then resuspended with 100µl of Tryptone salt and then plated on solid medium in TSA Petri dishes (bioMérieux). The dishes were incubated for 24 hours at 37°C before enumeration. The addition of PEG contributed to a 23.5% improvement in the precipitation yield (Table 7). Tab water 7: Enumeration on box of colonies of Pseudomonas aeruginosa after centrifugation with or without the presence of PEG Sample Precipitation solution Number of colonies Average 129 CFU total MgCl2 66 70 (54% of 129) 129 CFU total MgCl2 74 129 CFU total MgCl2+PEG 100 100 (77.5% of 129) 129 CFU total MgCl2+PEG 100 *: no fluorescence signal was detected after 50 amplification cycles.

Claims

1. Method for selective isolation of microorganisms of interest in a liquid biological sample comprising or likely to comprise, notably: • microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and • untargeted elements, i.e.: - untargeted cells whose cell membrane contains cholesterol, and - optionally, viruses with envelopes containing cholesterol, and - optionally mycoplasmas containing cholesterol, and - optionally, debris of microorganisms of interest and / or of untargeted cells, said method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol or the viral envelopes containing cholesterol or the membranes of mycoplasmas containing cholesterol, b) performing osmotic shock of the untargeted cells in order to lyse them specifically, c) adding an agent for precipitating the unlysed microorganisms of interest in solution in the sample, which is polyethylene glycol, d) adding a solution of at least one enzyme able to lyse the free nucleic acids (DNA and / or RNA) derived from the untargeted elements lysed in solution in the sample, allowing the microorganisms of interest to be obtained selectively.

2. Method for selective isolation, according to Claim 1, wherein the step consisting of adding an agent for precipitating the unlysed microorganisms of interest in solution in the sample is carried out in step a), after steps a) and b) or after step d).

3. Method for selective isolation of nucleic acids of interest in a liquid biological sample comprising or likely to comprise, notably: • microorganisms of interest whose cell membrane or capsid does not contain cholesterol, and • untargeted elements, i.e.: - untargeted cells whose cell membrane contains cholesterol, and - optionally, viruses with envelopes containing cholesterol, and - optionally mycoplasmas containing cholesterol, and - optionally, debris of microorganisms of interest and / or of untargeted cells, said method comprising the following steps: a) bringing the liquid biological sample into contact with a saponin formulation, in order to destabilize the cell membranes containing cholesterol and / or the viral envelopes containing cholesterol and / or the membranes of mycoplasmas containing cholesterol, b) carrying out osmotic shock of the untargeted cells in order to lyse them specifically, c) adding a precipitant of the unlysed microorganisms of interest in solution in the sample, which is polyethylene glycol, d) adding a solution of at least one enzyme able to lyse the free nucleic acids (DNA and / or RNA) derived from the untargeted elements lysed in solution in the sample, e) inactivating the enzyme added in step (d), and f) making accessible the nucleic acids of microorganisms of interest: - not degraded by the enzyme of step (d), and - not accessible by the action of steps a) and b).

4. Method for selective isolation of nucleic acids of interest in a liquid biological sample, according to Claim 3, wherein the step consisting of adding an agent for precipitating the unlysed microorganisms of interest in solution in the sample is carried out in step a), after steps a) and b) or after step d) or after step e).

5. Method of isolation according to any one of Claims 1 to 4, characterized in that the saponin is in a volume at least greater than or equal to the volume of the sample at the same concentration.

6. Method of isolation according to any one of Claims 1 to 5, characterized in that the final saponin concentration is above 0.02% and less than or equal to 20%.

7. Method of isolation according to any one of Claims 1 to 6, characterized in that the saponin consists of a triterpenoid.

8. Method of isolation according to any one of Claims 1 to 7, characterized in that the enzyme able to lyse the free nucleic acids is then inactivated: • chemically by adding EDTA and / or EGTA and / or DTT and / or β-mercaptoethanol and / or DEPC and / or guanidine and / or • physically by increasing the temperature between 40 and 100°C in the presence or in the absence of detergents, such as sodium dodecyl sulphate (SDS).

9. Method according to any one of Claims 1 to 8, characterized in that during the method, the pH is maintained in a range between 5 and 10, preferably between 6 and 9, by adding a solution that is: • basic if the pH is below 5, preferably below 6, • acidic if the pH is above 10, preferably above 9, so that the pH is within the range.

10. Use of a saponin formulation and of a solution of at least one enzyme able to lyse the free nucleic acids (DNA and / or RNA) and of at least one precipitant in solution, which is polyethylene glycol, for isolation of microorganisms of interest or of nucleic acids of microorganisms of interest, in a liquid biological sample.

11. Use according to Claim 10, characterized in that it uses a saponin formulation leading to a final concentration above 0.02% and less than or equal to 20%, and / or a precipitant at a concentration from 0.1 to 20%, and / or an enzyme able to lyse the free nucleic acids (DNA and / or RNA) containing between 500 and 20000 enzyme units.

12. Diagnostic test based on a method according to any one of Claims 1 to 9, or on a use according to any one of Claims 10 to 11.

13. Diagnostic kit for isolating the microorganisms of interest and / or for isolating the nucleic acids of microorganisms of interest in a liquid biological sample comprising or likely to comprise, notably, microorganisms of interest, untargeted cells and, optionally, enveloped viruses, mycoplasmas and / or debris of microorganisms of interest and / or of untargeted cells, said kit comprising: (a) a container, and (b) at least one saponin formulation, and (c) at least one solution of a precipitant, which is polyethylene glycol, (d) at least one enzyme able to lyse the nucleic acids, and (e) what is needed to detect the microorganisms of interest and / or the nucleic acids of microorganisms of interest.

14. Kit, according to Claim 13, characterized in that the kit further comprises: d') at least one acid solution and / or at least one basic solution, and / or f) EDTA and / or EGTA and / or DTT and / or β-mercaptoethanol and / or DEPC and / or guanidine and / or g) at least one detergent or an anionic agent.