Method of genetic analysis of a sample for detection of integrated microorganisms

Abstract

The present invention relates to a method for analysing the genetic material contained in a sample, especially a sample collected from the environment. The applications of the present invention more particularly relate to the detection and characterization of living microorganisms when they are present within a complex matrix in a sample. A method according to the invention allows the analysis of the genetic material present inside an integrated envelope, and comprises at least one step of treatment with at least one nuclease of a biological sample containing or liable to contain i) free nucleic acid and / or ii) nucleic acid included in an integrated envelope.

Description

[0001] METHOD FOR GENETIC ANALYSIS OF A SAMPLE FOR THE DETECTION OF INTEGRATED MICROORGANISMS

[0002] Field of invention

[0003] The present invention relates to a method for analyzing the genetic material contained in a sample, in particular a sample taken from the environment. The applications of the present invention relate more particularly to the detection and characterization of living microorganisms when they are present within a complex matrix in a sample.

[0004] The present invention is therefore in the field of analysis of genetic material.

[0005] State of the art

[0006] Samples containing biological material, particularly those taken from the environment, are commonly very rich in all kinds of components, including cells and cell debris, as well as so-called "free" nucleic acid, i.e., not contained in a cell, a viral envelope or any other envelope. However, it is often necessary to specifically distinguish, within such a complex sample, the presence of intact microorganisms that are potentially capable of reproducing. It is desirable to distinguish intact microorganisms from non-integrated and / or incapable microorganisms. The reproductive capacity of microorganisms is indeed conditioned by their integrity.

[0007] A method currently used to achieve the integrity of microorganisms is amplification by culture, particularly on Petri dishes. This technology, in addition to its cost and the time it entails, only partially achieves the desired objectives since not all intact microorganisms are necessarily cultivable, or do not thrive in the conditions or on the matrices used to cultivate them.

[0008] Another method is microscopic observation, carried out during a microscopic examination by an expert or by flow cytometry. However, the level of precision and reliability of these measurements does not reach the degree of precision of molecular biology methods and may depend on the experience of the person carrying out microscopic observation or cytometric detection. There is therefore a need for a method of analyzing a sample that can specifically distinguish intact microorganisms, said method being rapid, efficient and reproducible.

[0009] Statement of the invention

[0010] The inventors have now developed a method for analyzing the genetic material present in a sample, in which the genetic material contained within any intact envelope present in said sample is specifically detected. A method according to the invention therefore makes it possible to sensitively and specifically detect genetic material not parasitized by so-called "free" nucleic acid, i.e. not included in an intact envelope.

[0011] The inventors have also developed a method for modifying the genetic material present in a sample, in which the genetic material contained within any intact envelope present in said sample is specifically modified. A method according to the invention therefore also makes it possible to specifically modify genetic material not parasitized by so-called "free" nucleic acid, i.e. not included in an intact envelope.

[0012] A first step of the method according to the invention consists, during the preparation and extraction prior to genetic analysis by PCR, in digesting with a nuclease the nucleic acid molecules not enveloped in an intact membrane. In a sample taken from the environment, said free nucleic acids originate in particular from cells or viruses whose membrane is degraded. Said nucleic acids can also be contained in the spores of dormant microorganisms.

[0013] A method according to the invention leads to the degradation of free nucleic acids and those not included in an intact compartment, which has the advantage of eliminating, in complex matrices with a high concentration of microorganisms, free nucleic acids likely to hinder PCR detection due to congestion, and of discriminating between intact microorganisms and compartments, on the one hand, and non-integrated microorganisms and compartments, on the other hand.

[0014] It appears in fact that, in particular for applications of detecting the presence of microorganisms of any nature, whether eukaryotic, prokaryotic or viral, relevant information, in particular for evaluating the viability of said microorganisms, concerns more specifically the nucleic acid included inside intact envelopes.

[0015] Indeed, the reproductive capacity of microorganisms is conditioned by their viability, which is itself conditioned by the integrity of their genetic material, on the one hand, and the integrity of their structure, on the other.

[0016] A method according to the invention therefore comprises, prior to carrying out an analysis, in particular by PCR amplification, or a modification, in particular by means of a CRISPR / Cas9 type technique, a step of preparing a sample and adding to the prepared sample a nuclease chosen from nucleases active in the presence of a high saline concentration in the reaction medium, this high saline concentration being in fact necessary for PCR amplification. A method according to the invention comprises bringing into contact, under controlled reaction conditions, a nuclease and the genetic material present in the sample. The application of controlled reaction conditions leads to the degradation of the nucleic acid which is not included in an integral envelope such as a cell membrane, a spore envelope, a viral envelope or a synthetic capsule.On the other hand, during this step the nuclease does not cross, or only negligibly, the said cell membrane, spore envelope, viral envelope or synthetic capsule. The nucleic acids contained within the said envelopes are therefore preserved and can be analyzed or modified.

[0017] A method according to the invention combines the advantages of a molecular biology method such as PCR with the analytical relevance of microscopic observation or culture methods. A method according to the invention has, on the one hand, advantages of speed, sensitivity, versatility, reproducibility and low cost as well as, on the other hand, selective detection of the microorganism(s) of interest which are intact and viable.

[0018] The applications of the present invention are numerous and varied. Indeed, the invention finds a particular application in the detection of the presence of intact and / or viable microorganisms in a sample. Said microorganisms are preferably chosen from bacteria, yeasts and viruses. The invention also finds a particular application for genotyping. This is the case, for example, of pollen genotyping, genotyping of microorganisms capable of producing toxins or of being resistant to antibiotics, genotyping of protoplasts or gametes. Finally, the invention is applicable to the analysis of synthetic nucleic acid artificially encapsulated in synthetic membranes.

[0019] More particularly, the subject of the present invention is a method for analyzing the genetic material present in a sample, this method comprising a prior step of selecting an appropriate nuclease then a step of treating the sample of interest with said nuclease, under specific reaction conditions, prior to the amplification by PCR of at least one target nucleotide sequence.

[0020] The inventors propose a means for selecting in advance a nuclease capable of specifically degrading free nucleic acid while preserving the nucleic acid contained within an intact envelope, i.e. said nuclease is not active with respect to said nucleic acid present within an intact membrane. The present invention makes it possible to specifically detect the nucleic acid contained within any intact envelope, said envelope being not only a cell membrane but also a viral envelope, a spore envelope or a synthetic capsule. In particular, the present invention makes it possible to specifically detect intracellular nucleic acid, i.e. the nucleic acid contained within an intact cell and therefore considered, a priori, as viable.

[0021] Detailed description of the invention

[0022] In this description, the term "comprising" includes the term "including" and the expression "consisting of". For example, a composition comprising compound X means a composition comprising exclusively compound X or may also include another compound.

[0023] According to a first aspect, the subject of the invention is a method for analyzing or modifying the genetic material contained in an intact envelope, comprising at least the following steps: a) treatment with at least one nuclease of a first fraction of a biological sample containing or likely to contain i) free nucleic acid and ii) nucleic acid included in at least one intact envelope, then inactivation of said nuclease by any method known to the person skilled in the art, b) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, c) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, in a second fraction of the biological sample not subjected to treatment with said nuclease,d) comparison of the results obtained during steps b) and c) of detection of the target nucleotide sequence and degraded nucleic acid in said first and second fractions, e) selection of at least one nuclease capable of degrading the free nucleic acid while preserving the nucleic acid included in an intact envelope, f) treatment by said at least one nuclease of a test fraction of said biological sample, g) application, to at least one target nucleotide sequence of the nucleic acid present inside said intact envelope, of an analysis or modification method.,

[0024] The invention is thus of particular interest in determining, during step d), the operating conditions of at least one nuclease with the aim of detecting genetic material present inside an intact envelope. The method according to the invention thus makes it possible to adapt the operating buffer of the nuclease, the nuclease and the inactivation conditions of the latter. Thus, during step e), it is in particular all of these elements (operating buffer, nuclease and inactivation conditions) which are considered functional for the selection, as detailed below.

[0025] According to one embodiment, the biological sample comprises free nucleic acid and nucleic acid included in an intact envelope.

[0026] According to one embodiment, the biological sample used during steps a) and c) is a test biological sample, different from that of step f).

[0027] According to one embodiment, the target nucleotide sequence used during steps a) to c) is added in each l ère and 2 ème fraction of the biological sample in a known quantity. In particular, the target nucleotide sequence is added in enveloped form in an intact envelope and optionally also in free form. Alternatively, it is the natively present quantity of the target nucleotide sequence in each of the fractions that is analyzed. Of course, in this context the nucleotide sequence used in steps b) and c) is present in the nucleic acid included in an intact envelope.

[0028] According to one embodiment, the comparison in step d) makes it possible to select the operating conditions of the nuclease, the nuclease itself and the conditions of inactivation of this nuclease. Indeed, step d) makes it possible to determine whether the nuclease could have had an activity under the conditions carried out (for example difference in levels between steps b) and c)), in which case it will be selected during step e). In addition, step d) makes it possible to determine whether the inactivation of the nuclease was indeed carried out under the conditions (for example absence of total degradation of the target nucleotide sequence), in which case it will be selected during step e). According to a first embodiment of this first aspect, the subject of the invention is a method for analyzing the genetic material contained in an intact envelope,comprising at least the following steps: a) treatment with at least one nuclease of a first fraction of a biological sample containing or likely to contain i) free nucleic acid and ii) nucleic acid included in at least one intact envelope, then inactivation of said nuclease by any method known to the person skilled in the art, b) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, c) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, in a second fraction of the biological sample not subjected to treatment with said nuclease, d) comparison of the results obtained during steps b) and c) of detection of the target nucleotide sequence and degraded nucleic acid in said first and second fractions,e) selection of at least one nuclease capable of degrading the free nucleic acid while preserving the nucleic acid included in an intact envelope, f) treatment by said at least one nuclease of a test fraction of said biological sample, g) amplification by a PCR reaction of at least one target nucleotide sequence of the nucleic acid present inside said intact envelope, h) analysis of the genetic material present inside at least one intact envelope of said biological sample.,

[0029] According to a second embodiment of this first aspect, the subject of the invention is a method for modifying the genetic material contained in an intact envelope, comprising at least the following steps: a) treatment with at least one nuclease of a first fraction of a biological sample containing or likely to contain i) free nucleic acid and ii) nucleic acid included in at least one intact envelope, then inactivation of said nuclease by any method known to the person skilled in the art, b) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, c) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, in a second fraction of the biological sample not subjected to treatment with said nuclease,d) comparison of the results obtained during steps b) and c) of detection of the target nucleotide sequence and degraded nucleic acid in said first and second fractions, e) selection of at least one nuclease capable of degrading the free nucleic acid while preserving the nucleic acid included in an intact envelope, f) treatment by said at least one nuclease of a test fraction of said biological sample, g) modification of at least one target nucleotide sequence of the nucleic acid present inside said intact envelope by means of a CRIPR / Cas9 type technique.,

[0030] “Biological sample” means a sample containing or likely to contain biological material, i.e., any material containing genetic information and which is self-reproducing or reproducible in a biological system.

[0031] According to a particular embodiment, a method according to the invention is implemented from a biological sample chosen from: a sample from the environment or a sample of human or animal origin, and more particularly:

[0032] - a liquid sample, in particular a sample from the environment chosen from: groundwater, precipitation, surface water, ice and glacial melt, salt water, brine, estuary water, wastewater, water from industrial processes and drinking water,

[0033] - a solid sample, in particular a sample from the environment chosen from: a soil sample, a plant sample, in particular a plant or flower sample, a porous solid support, such as in particular a wipe,

[0034] - a gas sample, in particular a sample from the environment,

[0035] - a sample of human or animal origin, said sample being liquid or solid, such as in particular a biopsy or a sample of blood, urine, saliva or other bodily sample.

[0036] By “liquid sample” we mean a sample that is predominantly liquid in nature, possibly also containing particles or colloidal elements.

[0037] By “solid sample” we mean a sample of a predominantly solid nature.

[0038] The sample may comprise two or more targets, it may be purified or unpurified, and / or clarified or unclarified, prior to the method according to the invention. The sample may be a biological sample that has been treated for use in the methods of the invention. In certain embodiments, if the biological sample does not interfere with the methods of the invention, it may be used untreated (and / or unpurified).

[0039] The biological sample may include, but is not limited to, cells, tissues, blood products, bodily fluids, and viral material.

[0040] By "analysis of genetic material" we mean the characterization of the nucleic acid present in a sample.

[0041] The term "envelope" means eukaryotic and prokaryotic cell membranes, viral particles and any other spore-like envelope, as well as any type of synthetic envelope capable of containing nucleic acids. The term "integral envelope" means a container capable of delimiting an interior compartment isolated from the exterior of said compartment. Said envelope may in particular be of natural or synthetic origin. The term "genetic material contained in an integral envelope" means the genetic material included in the compartment delimited by an integral envelope. The term "envelope" means a container chosen from a cell envelope, including in particular a cell membrane and possibly a wall, a viral envelope, a spore envelope and a synthetic capsule.

[0042] The term "nucleic acid", "polynucleotide" or "oligonucleotide" means a polymer of nucleotide monomers or an analogue thereof, including double- and single-stranded polymers, deoxyribonucleotides, ribonucleotides, their alpha-anomeric forms, and similar polymers. Within said nucleic acid polymers, or nucleic acid chains, the monomers are notably linked by phosphodiester bonds, the term "phosphodiester bond" referring to phosphodiester bonds or to bonds comprising phosphate or analogues thereof, including associated counterions. The expression "polymer of nucleotide monomers" is also referred to as "nucleotide monomer chain" or "nucleotide chain".

[0043] By "target nucleotide sequence" is meant a particular sequence of a nucleic acid polymer to which a particular primer or probe is capable of specifically hybridizing. In a method according to the invention, one or more target nucleotide sequences may be present within a nucleic acid polymer.

[0044] The term "nuclease" means an enzyme whose main function, or the only known function, consists of the partial or total degradation of at least one type of nucleic acid. A nuclease mainly degrades the phosphodiester bonds of nucleic acid strands. Said nucleic acid is DNA, cDNA or RNA. According to a particular aspect, said nuclease is a deoxyribonuclease, or DNase, or DNase, which hydrolyzes DNA. According to another particular aspect, said nuclease is a ribonuclease or RNase, or RNase, which hydrolyzes RNA. The substrate of said nucleases is a single-stranded nucleic acid or a double-stranded nucleic acid. In a method according to the invention, said nuclease is an endonuclease or an exonuclease. In addition, said nuclease may be natural or recombinant. Said nuclease may be natural or recombinant. This definition excludes DNA polymerases capable of cleaving nucleotides.

[0045] According to a particular aspect, a method according to the invention comprises a step of treatment by at least one nuclease of a first fraction of a biological sample containing or likely to contain i) free nucleic acid and ii) nucleic acid comprised in at least one intact envelope, said step being carried out under specific reaction conditions likely to represent the reaction conditions encountered during the analysis of the genetic material of a sample taken from the environment. Said specific reaction condition is in particular chosen from:

[0046] - high saline concentration,

[0047] - a high concentration of tannins,

[0048] - a high concentration of components known for their inhibitory nature of PCR amplification reactions.

[0049] A step of treating a sample with at least one nuclease includes bringing together:

[0050] - an effective quantity of nuclease, expressed in nuclease units, and

[0051] - of the sample, at a temperature allowing nuclease activity and for a sufficient time to observe nuclease activity.

[0052] The person skilled in the art will easily be able to select the effective quantity of nuclease allowing degradation, at least partial and preferably total, of the free nucleic acid.

[0053] This step allows the selection of at least one nuclease likely to be active in the sample subject to the test method. The comparison carried out in step d) thus allows the selection of at least one nuclease during step e).

[0054] According to a particular aspect, to carry out this selection, the step of treatment with at least one nuclease of a first fraction of a biological sample containing or likely to contain i) free nucleic acid and ii) nucleic acid included in at least one intact envelope, is carried out under hypersalinity reaction conditions. This condition can in particular be recreated by the addition of a saline concentration of at least 10 mM, at least 20 mM, at least 50 mM, or 60 mM. The salt used for said saline addition is in particular sodium chloride (NaCl). A person skilled in the art will be able to choose any appropriate concentration of any type of salt.

[0055] In particular, a nuclease active in the presence of a high salt concentration indicates a nuclease whose reference catalytic activity is retained at least 70%, preferably at least 80% or at least 90% of its reference catalytic activity in a medium characterized by a high salt concentration.

[0056] In a method according to the invention, the nuclease concentration in the sample is defined in number of units (U), one nuclease unit being defined as the quantity of enzyme capable of completely degrading 1 pg of DNA in 10 minutes at 37°C in a reaction volume of 25 pL, the enzyme and the substrate being suspended in an appropriate buffer.

[0057] By "inactivation of said nuclease" is meant any known method of enzymatic inactivation, in particular by thermal inactivation, in particular in a dry bath at 65°C for 10 minutes, or by chemical inactivation, in particular by the addition of a conventional inactivation buffer, well known to those skilled in the art.

[0058] Genetic analysis of a sample is currently commonly carried out using techniques such as, in particular, the polymerase chain reaction (PCR), more particularly quantitative PCR (q-PCR) and digital PCR (dPCR). By "PCR" is meant any amplification method comprising at least one PCR step, more particularly a quantitative PCR (q-PCR) or digital PCR (dPCR) step. Preferably, a method according to the invention comprises a dPCR amplification step.

[0059] A method according to the invention is compatible with all digital PCR platforms on the market, in particular microdroplets and / or micro compartments. Among these, commercial equivalents such as the QIAcuity Digital PCR System - QIAGEN and QX ONE Droplet Digital PCR (ddPCR) System - Bio-Rad may be mentioned. Said PCR reaction is carried out in a multiplex digital PCR device.

[0060] The presence of a target nucleotide sequence is demonstrated according to methods well known to a person skilled in the art.

[0061] The measured signals include signals detected at a wavelength characteristic of a dye, fluorescent agents or radioactive agents. Signal detection includes any type of detection such as direct detection or a method of energy transfer between fluorescent molecules, such as FRET (Forster Resonance Energy Transfer).

[0062] The dyes are selected from all available dyes allowing the selective identification of a given nucleotide target. A person skilled in the art will readily know how to choose from all available dyes to select distinguishable dyes during the multiplex analysis. In particular, a person skilled in the art will know how to choose two or more different fluorophores having absorption and emission wavelengths well separated from each other.

[0063] Dyes can be attached to a primer or probe, with such attachment to a primer or probe being compatible with PCR amplification, or intercalate into the DNA, preferably double-stranded, where appropriate (intercalator dyes).

[0064] In a method according to the invention, the dyes are chosen from simple dyes (i.e. non-fluorescent) and fluorescent dyes (fluorophores). Fluorophores commonly used in FRET include fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA).

[0065] The radioactive agents are chosen from any agent usable in a multiplex dPCR method known to a person skilled in the art. These include phosphorus 32, tritium, technetium-99m, iodine 125 or any other element known in the art.

[0066] According to a second aspect, the invention relates to a method for analyzing or modifying the genetic material present inside an intact envelope, said method comprising at least the following steps: a) treatment with at least one nuclease of a biological sample containing, or likely to contain, i) nucleic acid included in at least one intact envelope and ii) nucleic acid not included in an intact envelope, said nuclease being chosen from: a DNAse chosen from:

[0067] - DNase TURBOTM (ref: AM2238 / 2239, Thermo Fisher Scientific, Waltham, MA, USA)

[0068] - DNase I-XT (ref: M0570, New England BioLabs, Beverly, MA)

[0069] - DNase I (ref: M0303, New England BioLabs, Beverly, MA) b) application to at least one target nucleotide sequence of the nucleic acid present inside said intact envelope, of an analysis or modification method.

[0070] According to a particular aspect, a method for analyzing the genetic material present inside an intact envelope, according to the invention, comprises at least the following steps: a) treatment with at least one nuclease of a biological sample containing, or likely to contain, i) nucleic acid included in at least one intact envelope and ii) nucleic acid not included in an intact envelope, said nuclease being chosen from: a DNAse chosen from:

[0071] - DNase TURBOTM (ref: AM2238 / 2239, Thermo Fisher Scientific, Waltham, MA, USA)

[0072] - DNase I-XT (ref: M0570, New England BioLabs, Beverly, MA)

[0073] - DNase I (ref: M0303, New England BioLabs, Beverly, MA) b) amplification by a PCR reaction of at least one target nucleotide sequence of the nucleic acid present inside said intact envelope, c) analysis of the genetic material of said biological sample, present inside an intact envelope.

[0074] According to another particular embodiment of this second aspect, the subject of the invention is a method for modifying the genetic material present inside an intact envelope, said method comprising at least the following steps: a) treatment with at least one nuclease of a biological sample containing, or likely to contain, i) nucleic acid included in at least one intact envelope and ii) nucleic acid not included in an intact envelope, said nuclease being chosen from: a DNAse chosen from:

[0075] - DNase TURBOTM (ref: AM2238 / 2239, Thermo Fisher Scientific, Waltham, MA, USA)

[0076] - DNase I-XT (ref: M0570, New England BioLabs, Beverly, MA)

[0077] - DNase I (ref: M0303, New England BioLabs, Beverly, MA) b) modification of at least one target nucleotide sequence of the nucleic acid present inside said integrated envelope by means of a CRIPR / Cas9 type technique. Preferably, in a method according to the invention, said envelope is chosen from: a cellular envelope, a viral capsule and a synthetic capsule.

[0078] The invention is applicable to the analysis of synthetic nucleic acid artificially encapsulated within any type of synthetic membrane.

[0079] According to a particular aspect, in a method according to the invention said nuclease is chosen from:

[0080] - a DNAse of the “DNase I-XT” type distributed by the company New England BioLabs.

[0081] According to a particular aspect, a method according to the invention comprises an additional step in which the sample prepared and treated with a nuclease is diluted prior to the PCR analysis itself. The dilution of the sample is preferably at least a factor of 10, preferably a factor of 50, more preferably a factor of 100.

[0082] According to a particular aspect, at least two different dilutions of the sample are analyzed by PCR. The advantage of this dilution step is to provide a limiting dilution of the sample, in order to promote the detection of a significant co-occurrence of the presence of several microorganisms in the same sample compared to the detection of a co-occurrence of several microorganisms which would be purely statistical. A second advantage of this particular aspect of a method according to the invention consists in providing at least two results for the genetic analysis of the same sample, and therefore in allowing a more complete analysis of said sample. By "at least two different dilutions" is meant two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen dilutions, or more depending on the needs of the genetic analysis.

[0083] According to a particular aspect of a method according to the invention, the amplification step by a PCR reaction is preceded by a step of lysis of the envelopes likely to be present in said intact sample. A membrane lysis method can easily be implemented depending on the nature of the envelope whose lysis is desired. In particular, said lysis step will be carried out by adding a lysis buffer, well known to those skilled in the art, and by providing mechanical pretreatment to the sample; this pretreatment may in particular consist of a step of grinding or friction of the sample in the presence of glass beads. This thermal pretreatment leads to the weakening of resistant membranes, in particular the membranes of spores and the membranes of certain bacteria.

[0084] The kits used are:

[0085] - NucleoMag DNA / RNA Water kit, MWA1 Lysis Buffer (ref: 744220.4, Macherey Nagel, Germany)

[0086] - NucleoMag Pathogen kit, NPL1 Lysis Buffer (ref: 744210.4, Macherey Nagel, Germany)

[0087] - NucleoMag Tissue kit, manufacturer of Lyse Tl (ref: 744210.4, Macherey Nagel, Germany).

[0088] A method according to the invention is compatible with all PCR platforms on the market, in particular digital PCR (dPCR), in particular in microdroplets and / or micro compartments. Among these, commercial equivalents such as the QIAcuity Digital PCR System - QIAGEN and QX ONE Droplet Digital PCR (ddPCR) System - Bio-Rad may be mentioned. According to one aspect of the invention, said method comprises a PCR reaction carried out in a digital PCR (dPCR) device.

[0089] According to a more particular aspect, a method according to the invention comprises a multiplex PCR reaction.

[0090] According to a particular aspect, the invention relates to the use of a method according to the invention for analyzing genetic material for the detection of at least one microorganism in a sample.

[0091] According to another particular aspect, the invention relates to the use of a method according to the invention for analyzing genetic material for detecting the co-occurrence of at least two microorganisms in the same sample.

[0092] According to a particular aspect, the invention relates to the use of a method according to the invention for analyzing the genetic material present in a sample, for detecting the co-occurrence of at least two microorganisms in the same sample.

[0093] According to a particular aspect, the invention relates to the use of a method according to the invention for analyzing the genetic material present in a sample for genotyping. By "genotyping" is meant the determination of a genetic variation at a given position of the genome considered.

[0094] Other advantages and characteristics will appear upon examination of the detailed description of a non-limiting embodiment, and the attached drawings.

[0095] Figure 1 of Example 1 represents the scatter diagrams of the results obtained under “classical” reaction conditions of DNAse action during dPCR amplification of samples prepared in vitro, lines A, B and C of the figure correspond respectively to the following conditions:

[0096] - A: E. coli culture,

[0097] - B: DNA extract from E. coli and

[0098] - C: E. coli culture supplemented with an E. coli DNA extract.

[0099] From left to right, the results show untreated samples (left column), treated with DNAse I (center column), or treated with DNAse XT (right column). For each figure, the number of positive partitions (Part. +), negative partitions (Part. -), and the detection threshold are indicated.

[0100] Figure 2 of Example 1 represents the scatter diagrams of the results obtained under reaction conditions of DNAse action in high salinity, with:

[0101] - line A: an extract of E. coli DNA, and

[0102] - line B: an extract of E. coli culture supplemented with an extract of E. coli DNA.

[0103] From left to right, the results show untreated samples (left column), treated with DNAse I (center column), or treated with DNAse XT (right column). For each figure, the number of positive partitions (Part. +), negative partitions (Part. -), and the detection threshold are indicated.

[0104] Figure 3 of example 2 shows schematically the steps of a method according to the invention.

[0105] It is understood that the embodiments which will be described subsequently are in no way limiting. In particular, it is possible to imagine variants of the invention comprising only a selection of characteristics described subsequently isolated from the other characteristics described, if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention from the state of the prior art. The present invention will be better understood by reading the following examples, which are given to illustrate the invention and not to limit its scope.

[0106] EXAMPLES

[0107] Example 1: In vitro detection of intact microorganisms

[0108] Materials and methods

[0109] Samples are prepared from an in vitro culture of E. coli according to different reaction conditions presented in Table 1 below: [TABLE 1]

[0110] The Escherichia coli culture is carried out from a glystock, it is a 14H culture at 37°C with shaking at 300 rpm in LB (lysogeny broth) media. The extraction is carried out with the NucleoMag DNA / RNA Water kit (ref: 744220.4, Macherey Nagel, Germany) from a test sample of 100 pL of the culture.

[0111] The condition "E. coli bacterial culture" corresponds to 100 pL of said culture, the condition "E. coli DNA extracts" corresponds to DNA extracted from 100 pL of said culture, the condition "E. coli bacterial culture + E. coli DNA extracts" corresponds to 100 pL of the culture supplemented with DNA extracted from 100 pL of the culture. The samples are stored at 4°C pending further processing.

[0112] If necessary, the samples are supplemented with a nuclease under the following conditions:

[0113] - addition of DNAse I or DNAse I-XT from the supplier NEB (New England BioLabs), according to the supplier's recommendations, i.e. 2u of DNase I-XT or DNase I, Buffer 10X in a final volume of 100 pL. The reaction conditions are: either the standard conditions recommended by the supplier, or the high saline conditions. Either:

[0114] - DNase I-XT Reaction Buffer (10x) + 2 units DNase I + qsp 100 pL H2O Nuclease free. Incubate for 15 min at 37°C and - DNase I-XT Reaction Buffer (10x) + 2 units DNase I + qsp 100 pL H2O Nuclease free. Incubate for 10 min at 37°C, then add 1 pL of 0.5M EDTA then incubate for 10 min at 75°C then add 1 pL of a 6M NaCI stock solution.

[0115] The samples are then analyzed by PCR. The following products and kits are used: QIAcuity Nanoplate 26K 24 well (Ref. ID: 250001) and QIAcuity Probe PCR Kit Qiagen, (Ref. ID: 250102). In the PCR mix, the forward and reverse primers are present at a final concentration of 450 nM, the probe 5'-HEX- CCTGCCGCGTTGGCAATGTCGAGT-BHQ-1-3' (SEQ ID No. 1) is present at a final concentration of 125 nM. The nucleotide sequences, amplification conditions and signal detection conditions are respectively described in Tables 2, 3 and 4 below.

[0116] [Table 2]

[0117] [Table 3] [Table 4]

[0118] Results

[0119] The results are analyzed by QIAcuity Software Suite, they are expressed in number of copies per microliter of sample and presented in the following Table 5:

[0120] [Table 5]

[0121] Figure 1 represents the scatter diagrams of the results obtained under “classical” reaction conditions of DNAse action with: - line A: an extract of E. coli culture,

[0122] - line B: an extract of E. coli DNA, and

[0123] - line C: an extract of E. coli culture supplemented with an extract of E. coli DNA.

[0124] From left to right, results show untreated (left column), DNAse I-treated (center column), or DNAse XT-treated (right column) samples.

[0125] In the case of the E. coli culture extract (Figure 1 row A), the experimental results show that DNAse I did not cleave the DNA while DNAse XT cleaved part of the culture. Bacterial mortality is observed, which led to free DNA that was cleaved by DNAse XT. In the case of the E. coli DNA extract (Figure 1 row B), the experimental results show that DNAse I partially cleaved the DNA while DNAse XT almost completely cleaved the DNA. In the case of the E. coli culture extract supplemented with an E. coli DNA extract (Figure 1 row C), the experimental results show that DNAse I did not cleave the DNA while DNAse XT cleaved part of the free DNA.

[0126] In Figure 2, lines A and B represent respectively the scatter diagrams of the results obtained under reaction conditions of DNAse action in high salinity, with a concentration of 60 mM of NaCl with:

[0127] - line A: an extract of E. coli DNA, and

[0128] - line B: an extract of E. coli culture supplemented with an extract of E. coli DNA.

[0129] From left to right, results show untreated (left column), DNAse I-treated (center column), or DNAse XT-treated (right column) samples.

[0130] In the case of the E. coli DNA extract (Figure 2 line A), DNAse I is sensitive to the presence of NaCl at a concentration of 60 mM, with the presence of a “rain” cluster, whereas DNAse XT cleaved almost all of the free DNA.

[0131] In the case of the E. coli culture extract supplemented with an E. coli DNA extract (Figure 2 line B), DNAse I did not cleave the DNA while DNAse XT cleaved some of the free DNA. The results also show the uncleaved DNA corresponds to the intact E. coli bacteria. Mortality was observed in the bacterial culture.

[0132] Conclusion

[0133] These results show that under control conditions on a simple matrix, i.e. a “non-environmental” sample, with or without NaCl, DNase I-XT is active and presents a higher percentage of free DNA digestion than that of DNase I.

[0134] Example 2: Detection of the presence of the bacterial strain Microthrix in environmental samples

[0135] Materials and methods

[0136] Figure 3 shows a schematic of a method according to the invention, in which the matrix comprises free nucleic acid and microorganisms. The addition of a high-tolerance DNAse to this medium leads to the specific degradation of the free nucleic acid, without acting on the nucleic acid enveloped in an intact microorganism. After treatment with DNAse, the nucleic acid is extracted from the environment and then amplified by dPCR.

[0137] Environmental samples constitute a complex matrix, including PCR inhibitors, including a high concentration of salts and tannins and the presence of free nucleic acids.

[0138] Samples are collected at various points in a wastewater treatment plant to detect the presence of the Microthrix bacterial strain in its natural environment. The "WWTP inlet" sample is collected before treatment in the wastewater treatment plant (WWTP). The "digester" sample is collected during the anaerobic treatment of sludge-type waste. The "recirculating sludge" sample is collected from the sludge after treatment in the digester. Samples are stored at 4°C pending further processing.

[0139] The samples, either sewage treatment plant sludge or sewage treatment plant wastewater, are then pretreated as indicated below.

[0140] Sewage sludge is treated as follows from the raw sample received, with a volume of up to one litre or one kilogram:

[0141] - Homogenize by turning 10 times

[0142] - Take 200 pL in a 2 mL tube

[0143] - Centrifugation at room temperature (RT): 15 min-5000 g

[0144] - Removal of the supernatant

[0145] - Addition of 20 pL DNase I-XT Reaction Buffer (10x) + 2 pL (4units) DNase I-XT + 178 pL H2O Nuclease free

[0146] - Resuspend the pellet then incubate for 15 min at 37°C. Centrifuge for 5 min at 5000 g then remove the supernatant

[0147] - Addition of 2 glass beads (diameter 6 mm)

[0148] - GENOGRINDER cycle: 1 cycle of 30 sec at 1400 cpm

[0149] - Centrifugation at RT: 30s-5000 g

[0150] - Add 200 pL PBS lx

[0151] - Vortex: 5 min-1500 rpm

[0152] - Centrifugation at RT: 5 min-500 g

[0153] - Transfer the supernatant into an identified 1.5 mL tube.

[0154] The sample is ready for extraction. For the wastewater treatment plant sludge matrix: Add 20 pL DNase I-XT Reaction Buffer (10x) + 2 pL (4 units) DNase I-XT + 178 pL H2O Nuclease free. The pellet is resuspended then incubated for 15 min at 37°C, centrifugation for 5 min at 5000 g and removal of the supernatant are carried out.

[0155] Wastewater from the treatment plant is treated as follows, from the raw sample received (up to 1 L):

[0156] - 10 flips

[0157] - 15 mL sample

[0158] - Centrifugation at RT: 15 min-3234 g

[0159] - Removal of the supernatant

[0160] - Add 20 pL DNase I-XT Reaction Buffer (10x) + 2 pL (4 units) DNase I-XT + 178 pL H2O Nuclease free. Resuspend the pellet then incubate for 15 min at 37°C. Centrifuge.

[0161] The sample is ready for extraction.

[0162] For the wastewater matrix: add 20 pL DNase I-XT Reaction Buffer (10x) + 2 pL (4 units) DNase I-XT + 178 pL H2O Nuclease free. Resuspend the pellet then incubate for 15 min at 37°C. The pellet is resuspended then incubated for 15 min at 37°C, centrifugation for 5 min at 5000 g and removal of the supernatant are carried out.

[0163] The samples are then analyzed by PCR. The following products and kits are used: QIAcuity Nanoplate 26K 24 well (Ref. ID: 250001) and QIAcuity Probe PCR Kit Qiagen, (Ref. ID: 250102). In the PCR mix, the forward and reverse primers are present at a final concentration of 450 nM, the probe 5'-FAM- TGAAATCTCAGGGCCCAACCCTGAGC-BHQ-1-3' (SEQ ID No. 4) is present at a final concentration of 125 nM.

[0164] The nucleotide sequences, amplification conditions and signal detection conditions are respectively described in Tables 6, 7 and 8 below.

[0165] [TABLE 6] [TABLE 7]

[0166] [TABLE 8]

[0167] Results are analyzed by QIAcuity Software Suite and Easydata. Results are expressed as the number of copies per liter of sample. Table 9 shows the results by collection date.

[0168] [TABLE 9]

[0169] Table 10 presents the results by sample type.

[0170] [TABLE 10]

[0171] The experimental results show a difference observed between the recirculation sludge and the digester, between the samples treated with DNAse and the untreated samples. In the WWTP inlet samples from 22-09 and 27-09, DNAse would cleave all the free DNA and there would be no intact bacteria. For the sample from 4-10-22, there would be only intact bacteria. The samples not treated with DNAse are more concentrated than the samples treated with DNAse, the difference between the two groups of samples is 1 to 2 log. The “Microthrix without DNAse” samples contain free DNA and intact bacteria. The “Microthrix with DNAse” samples contain intact bacteria.

[0172] Conclusion

[0173] The results show a difference in quantification with or without DNase I-XT treatment. These results are consistent with the presence of free DNA in the raw sample, eliminated by DNase I-XT treatment.

Claims

CLAIMS 1. Method for analyzing or modifying the genetic material present inside an intact envelope, comprising at least the following steps: a) treatment with at least one nuclease of a first fraction of a biological sample containing or likely to contain i) free nucleic acid and / or ii) nucleic acid included in an intact envelope, b) detection of a target nucleotide sequence and nucleic acid degraded by said treatment in said first fraction, c) detection of said target nucleotide sequence and said nucleic acid degraded by said treatment in said first fraction, in a second fraction of the biological sample not subjected to treatment with said nuclease, d) comparison of the results obtained during steps b) and c) of detection of the target nucleotide sequence and degraded nucleic acid in said first and second fractions,e) selection from the comparison obtained in step d) of at least one nuclease capable of degrading the free nucleic acid while preserving the nucleic acid included in an intact envelope, f) treatment by said at least one nuclease of a test fraction of said biological sample, g) application to at least one target nucleotide sequence of the nucleic acid present inside said intact envelope, of an analysis or modification method., 2. Method according to claim 1, in which step g) consists of an amplification by a PCR reaction of at least one target nucleotide sequence of the nucleic acid present inside said integral envelope and is followed by a step of analyzing the genetic material of said biological sample.

3. Method according to claim 1, in which step g) consists of a modification of at least one target nucleotide sequence of the nucleic acid present inside said integrated envelope by means of a CRIPR / Cas9 type technique.

4. Method according to one of the preceding claims, in which said integral envelope is chosen from: a cellular envelope, a viral capsule and a synthetic capsule.

5. Method according to one of the preceding claims, characterized in that said nuclease is: a DNAse of the “DNase I-XT” type from the distributor New England BioLabs.

6. Method according to any one of the preceding claims, characterized in that the amplification step by a PCR reaction is preceded by a dilution of the sample, said dilution being at least a factor of 10, preferably a factor of 50, more preferably a factor of 100.

7. Method according to any one of the preceding claims, characterized in that the step of amplification by a PCR reaction is preceded by a step of destruction of said intact envelope.

8. Method according to any one of the preceding claims, characterized in that the amplification step by a PCR reaction is carried out in a digital PCR (dPCR) device.

9. Method according to any one of the preceding claims, characterized in that said PCR reaction is multiplexed.

10. Use of a method according to any one of the preceding claims for detecting the presence of at least one microorganism in a sample.

11. Use of a method according to the preceding claim for: the detection of the co-occurrence of at least two microorganisms in the same biological sample, for genotyping and / or for the analysis of genetic material included in a synthetic envelope.

12. Use of a method according to any one of claims 1 to 5 for the modification of genetic material included in a synthetic envelope.