FR2827873A1 - Process for detecting presence of pathogenic organisms in water sample, by concentrating pathogenic organisms, opening cells of pathogenic organisms, amplifying nucleic acid and detecting DNA and/or RNA - Google Patents

Abstract

Detecting the presence of pathogenic organisms in water sample, involves concentrating pathogenic organisms, breaking to open cells of pathogenic organisms, concentrating DNA and/or RNA, amplifying nucleic acid and detecting DNA and/or RNA by qualitatively and/or quantitatively. The process is controlled fully automatically by using a control logic system. An Independent claim is included for a device for detecting the presence of pathogenic organisms in a water sample. The device comprises a unit (1) for concentrating the pathogenic organisms, a unit (2) for breaking open cells from the pathogenic organisms, a unit (3) for concentrating DNA and/or RNA, a unit (4) for amplifying nucleic acids, a unit (5) for qualitatively and/or quantitatively detecting DNA and/or RNA and a control logic system (6) which controls fully automatically the sequential treatment steps carried out by units (1) to (5), pumping units (7), automatic sampling and distribution units (8), and a central control system (9).

Description

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 The present invention relates to the general technical field of the detection of pathogenic organisms in water.

 In particular, the present invention relates to a fully automated method for detecting the presence of pathogenic organisms in a water sample. The invention also relates to a device for detecting the presence of pathogenic organisms in a water sample for the implementation of this process.

 The presence of pathogens in water supply networks, such as drinking water or industrial water networks, poses real risks to the health of human beings. Most water supply systems thus contain pathogenic organisms such as viruses, bacteria, protozoa, fungi, amoebae or worms which are capable of causing various diseases in humans. Among these diseases, we can cite among others cholera, typhoid fever, diarrhea, dysentery, legionellosis or even gastroenteritis.

 Among the pathogenic organisms responsible for such diseases, there may be mentioned more particularly the protozoa Cryptosporidium (parvum) and Giardia Lamblia which both originate from human and animal faecal waste and which are a major cause of gastroenteric diseases; the protozoa Toxoplasma gondii and Naegleria fowleri which also originate from human and animal faecal waste and which cause various serious diseases; Cyclospora which causes diarrhea; coliform bacteria, notably Escherichia coli, which are found throughout the biosphere and whose detection is important because they are considered, not as typical pathogens, but as organisms indicative of water contamination, since they only cause disease at very high concentrations. For the sake of simplification in the context of the present invention, the term “pathogenic organisms” will be used to group together all the organisms which are indicators of water contamination and pathogenic organisms actually responsible for diseases in humans. Mention may also be made of pathogenic organisms responsible for diseases, the Salmonella bacteria, the

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 S. enteridis, S. enterica, S. thyphi and S. paratyphi serotypes, which are responsible for 70% of foodborne bacterial diseases, including typhoid and paratyphoid fevers, and which cause a large number of deaths; Helicobacter pylori which is an organism linked to the origin of at least 75% of all stomach ulcers and to two types of stomach cancer, and which is also the cause of a large number of deaths; Legionella, which is found in natural areas as well as in water heating systems, which causes Legionnaire's disease; as well as viruses of human and animal origin, and in particular hepatitis A viruses, Norwalk type viruses, rotaviruses, adenoviruses, enteroviruses and rheoviruses.

Norwalk viruses notably cause sporadic and epidemic gastrointestinal diseases with diarrhea. We see about 200,000 cases a year appear in the United States.

 Given the presence of various pathogens in all water supply networks, these must be checked and possibly disinfected, before the water from these networks can be considered potable. With regard to wastewater or cooling water supply networks as well as energy production networks, in particular those for nuclear power production, it is sometimes necessary to analyze the water and even the disinfect before being able to reuse or reject it in the surrounding environment.

 The detection of the presence of pathogenic organisms, in particular the detection of highly pathogenic organisms with slow growth, which are the most difficult to detect, thus represents a major challenge in the fields of water treatment, distribution of the water as well as water quality control. The traditional pathogen detection techniques developed up to now have various drawbacks: they are not used continuously or in real time, they are of low sensitivity towards pathogenic organisms and they prove to be long and costly in terms of equipment and personnel.

 A first known technique uses the enumeration of the microorganisms which develop after culturing the sample on different selective nutritive media. This technique is simple but presents significant risks of errors and artifacts (low specificity of the morphological criteria, absence of development of microorganisms with very slow growth, exponential growth

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 on the nutrient medium of microbial strains which are actually in the latent growth phase in the sample). Its response time is, moreover, generally greater than 24 hours.

 A second technique, using the measurement of one or more enzymatic activity (ies), allows rapid quantification of populations of living microorganisms (cultivable microorganisms and / or microorganisms in viable but non-cultivable form) (WO 90/100983). This technique allows in particular the monitoring of a set of populations, but does not allow access to a very fine level of specificity.

 A third technique, using immunological probes, has a response time frequently greater than 24 hours, and often lacks sensitivity and specificity (artefacts due to cross-reactions can be observed).

 Other recent techniques use specific oligonucleotide probes, generally labeled, so as to allow their detection after hybridization to their targets. Two main types of oligonucleotide probes have been developed: probes targeting DNA (or mRNAs which correspond to them), and probes targeting rRNA (ribosomal RNA). However, DNA or mRNA probes, although potentially very specific, have the disadvantage of being insensitive due to the low number of DNA copies (or mRNA) per microbial cell.

 There was thus a need to develop a method and a device for detecting the presence of pathogenic organisms in a water sample, said method and device not having the drawbacks of the techniques of the prior art and allowing thus performing online detection of several pathogenic microorganisms in water in a very short time (less than 6 hours), at low cost and with high sensitivity and specificity, making it possible to reach the detection level of a organism in 100 ml of water sample analyzed, thus meeting drinking water quality standards.

 The present invention fulfills this need. The Applicant has thus surprisingly discovered that this could be achieved using a process and a device, managed in a fully automatic manner, usable at scale.

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 industrial, preferably based on the DNA or RNA amplification technique by a polymerase chain reaction (PCR) or a similar technique.

 The method according to the present invention is thus fully automated and does not contain any manual steps. It is based on the detection of DNA or RNA sequences, specific for certain pathogenic microorganisms. The automation of certain unit steps has already been described in the prior art, but the automation of all the sequential steps of the method according to the present invention had never been envisaged until then. Thus, the extraction on a solid support and the purification of nucleic acids, present in a liquid sample, have thus already been automated and have been described in patent applications WO 00/75623 and WO 97/10331. Similarly, the automation of the amplification of nucleic acids carried out by a polymerase chain reaction (PCR) as well as the detection of organisms by fluorescence had already been envisaged in patent application WO 00/33962. However, the problems encountered in implementing the automation of the five sequential steps of the method according to the present invention had thus never been overcome until now.

 The present invention thus relates to a method for detecting the presence of pathogenic organisms in a water sample comprising the following sequence of steps: a concentration of pathogenic organisms, an opening of the cells of pathogenic organisms, a concentration of DNA and / or RNA of cells, at least one amplification of nucleic acids, and a qualitative and / or quantitative detection of DNA and / or RNA representative of the contamination of water by said pathogenic organisms; characterized in that all of the sequential steps of the process are managed entirely automatically using a control logic.

 The water analyzed in the detection method according to the present invention advantageously comes from drinking water networks, industrial water networks or

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 waste water, cooling water networks, energy production networks, in particular nuclear energy.

 Advantageously according to the present invention, the pathogenic organisms detected are preferably bacteria, viruses, fungi and / or protozoa.

 Advantageously according to the present invention, the initial volume of water sample is greater than 50 ml, preferably between 100 ml and 10 liters, even more preferably from 2 to 3 liters.

 According to the present invention, an amplification of nucleic acids is necessary in the detection method in order to be able to detect at least one pathogen, and preferably from 1 to 5 pathogens, in the sample of water analyzed.

 Advantageously according to the present invention, the amplification of nucleic acids is carried out by a polymerase chain reaction (PCR) in real time.

PCR is a commonly used amplification technique. The PCR technique (Rolfs et al., 1991) requires the choice of pairs of oligonucleotide primers flanking the fragment which must be amplified. The general principles and the conditions for carrying out the amplification of nucleic acids by PCR are well known to those skilled in the art and are described in particular in US Patents 4,683,202, US 4,683,195 and US 4,965,188.

 Other similar nucleic acid amplification techniques (DNA and / or RNA) can be advantageously used as an alternative to PCR (PCRlike) using pairs of primers of nucleotide sequences. The term “PCR-like” is intended to denote all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or else in which the labeling systems have been amplified, these techniques being of course known to those skilled in the art. . Mention may in particular be made of the SDA technique (Strand Displacement Amplification) or strand displacement amplification technique (Walker et al., 1992), the TAS technique (Transcription-based Amplification System) described by Kwoh et al. (1989), the 3SR (Self-Sustained Sequence Replication) technique described by Guatelli et al. (1990), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. (1991), the TMA technique (Transcription

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 Mediated Amplification), the LCR (Ligase Chain Reaction) technique described by Landegren et al. (1988), the RCR (Repair Chain Reaction) technique described by Segev (1992), the CPR (Cycling Probe Reaction) technique described by Duck et al.

(1990), the Q-beta-replicase amplification technique described by Miele et al.

(1983). Some of these techniques have since been perfected. Thus, according to the present invention, specific detection of pathogens can be carried out using such techniques of amplification of nucleic acids at high speed.

 By comparing the amplification technique by PCR or by a similar technique such as NASBA with traditional techniques for the detection of pathogens such as cell culture, the Applicant has discovered that the time required for the test from a few weeks or a few days to a few hours. In addition, the amplification by PCR or the like is easy to carry out and the initial costs as well as the subsequent costs for the implementation of the PCR prove to be much lower than the costs generated for the implementation of the cell culture techniques. In addition, PCR can be used to identify a specific form of the pathogens found in water. It can thus be used to detect the infectious state of an organism, proving the presence or absence of DNA or RNA specific for the pathogen. The PCR amplification according to the invention also makes it possible to obtain a high sensitivity method, advantageously greater than 1 picogram, and even more advantageously greater than a few femtograms of nucleic acids.

 The Applicant has also discovered that the use of amplification by PCR (or the like) followed by specific detection in situ, in real time, for example by fluorescence, provides analysis data relating to the detection and identification of organisms faster and more precisely than any other biochip-based detection system. Biochips allow a much larger number of pathogenic species to be detected than that detected using online PCR but, on the basis of known pathogens (which can be more effectively tested as a group), the Real-time PCR is much more efficient and gives much better specificity than standard hybridized biochips. In addition, a hybridization of chips, which allows contacting of the microorganisms with a DNA and / or RNA probe, takes several hours, up to

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 Often include an overnight incubation, which is significantly longer than amplifying DNA or RNA in real time.

 To carry out the amplification of nucleic acids, preferably by PCR, according to the present invention, a wide variety of primers can be used, some of them already being available in databases or in the literature, some others having already been developed for specific organizations.

 According to a particular embodiment of the present invention, a "nested" PCR is used, by dividing the PCR into two stages, in order to obtain maximum sensitivity with respect to the pathogens which it is sought to detect in the water. The first PCR is then made with the entire sample (typically several hundred micro liters with only a few DNA molecules) using a set of degenerate primers using a standard thermal cycle device.

After amplification for a few cycles (for example 10 to 20 cycles) at high temperature (75 to 100 C), the fluid which then contains at least several thousand copies of each DNA can be divided into a number of small vials in a second PCR apparatus, using an automatic sampling and distribution means such as a robot. A second set of primers is then added to each flask and a second amplification of nucleic acids is then carried out while carrying out on-line detection, for example by fluorescence. The first amplification will thus be able to be used to amplify the nucleic acids of groups of pathogens using universal primers which are degenerate, while the second amplification will make it possible to precisely determine the species present in the sample analyzed at using primers specific to each species that one seeks to detect. In this particular embodiment of the present invention, the detection is carried out during the carrying out of the second PCR; no detection is carried out during the first PCR which is limited to a role of amplification of the nucleic acids.

 While in the medical field, it involves analyzing blood samples from a few microliters to a few milliliters, in research and monitoring relating to the environment, samples of volumes up to several tens of liters are usually analyzed. However, the techniques

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 direct opening of the cells of the organisms cannot be carried out on such large volumes, and the pathogenic organisms which it is sought to detect according to the method of the present invention must be concentrated at volumes of less than 100 ml before be able to start further treatment. In addition, drinking water quality standards require proof of the absence of an organism in 100 ml of the sample of water analyzed. The difficulty of concentrating pathogenic organisms from samples of the order of a liter of water is to lose as few organisms as possible, while reducing the volume of the sample to a volume of the order of a hundred ml.

 Advantageously according to the present invention, the concentration of pathogenic organisms is carried out by precipitation, co-precipitation or precipitation by inclusion. Such a concentration of organisms from sample volumes of the order of a liter thus makes it possible to have a highly sensitive process and to comply with the standards imposed on the quality of the water. In fact, the larger the sample volume, the more the sensitivity is increased and the more precise the analysis data. In addition, the detection method according to the present invention makes it possible to detect the presence of less than 10 different species in initial volumes of samples from 100 ml to 10 liters, preferably 2 to 3 liters. In addition, while competitive instruments use either filtration or magnetic bead capture as the first stage of concentration, precipitation by inclusion is more effective in terms of trapping organisms, more economical and allows to process highly contaminated samples. In the context of the present invention, the step of concentrating the pathogenic organisms comprises the automatic taking of the analyzed water samples.

 Even more advantageously according to the present invention, the concentration of pathogenic organisms is carried out in the presence of carbonates of bivalent ions, advantageously chosen from the group consisting of calcium carbonate, strontium carbonate and barium carbonate. The reaction medium is brought to a relatively high temperature, preferably of the order of 40 to 60 ° C., so as to allow the rapid formation, in a few minutes, of a precipitate containing various pathogenic agents by simple decantation and without adding additional gravitational force. In order to achieve effective decantation, reagents such as

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 carbonates of bivalent ions are not added all at once to the reaction medium, but are added several times. After formation of the precipitate, on which the various pathogens of the water sample are absorbed, the supernatant liquid is removed and the precipitate is dissolved in an acid such as formic acid. The reaction medium containing the dissolved precipitate is then neutralized using a buffering agent.

 Advantageously according to the present invention, the step of opening the cells, which follows that of the concentration of pathogenic organisms, is carried out by grinding on agitated pearls. By the term "cell opening" is meant in the sense of the present invention a rupture or lysis of the cells of pathogenic organisms aimed at releasing the content, and in particular the nucleic acids, of these cells. The operating conditions of this step is adapted so as to avoid any alteration of the nucleic acids The cell opening step according to the present invention is carried out using volumes of analyzed samples of the order of 10 to 100 ml, preferably 50 ml , and was preferably designed to take place in a semi-continuous system using beads stirred by a strong source of energy to break the cells of organisms by means of shocks and direct impacts. invention are retained in the cracking chamber (cell opening chamber), whereas the ruptured cells, following the shocks generated by the particles, and the acids nucleic acid thus released from the cells leave the cracking chamber, thus making it possible to operate the grinding of the pearls in a semi-continuous manner.

 Advantageously according to the present invention, the pearls are agitated by ultrasound, by mechanical agitation or by mechanical vibration, preferably for approximately ten minutes. The rise in temperature, created during agitation and shock of the particles, also seems to contribute to the opening efficiency of the cells. The addition of chemical agents or enzymes such as proteinases can also promote cracking of the cells, in particular by digesting the cell walls.

 Advantageously according to the present invention, during the opening of the cells or following the opening of the cells and before the concentration of DNA and / or RNA, are added to the reaction medium containing the cells, preferably with help from

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 pumps and valves, at least one chaotropic agent, advantageously a guanidine salt, such as guanidine thiocyanate, and at least one adsorption modifying agent, advantageously an alcohol such as ethanol, in order to facilitate the extraction of DNA and / or RNA of cells and in order to inactivate nucleases, which are enzymes capable of destroying nucleic acids. The guanidine salt, such as guanidine thiocyanate, is preferably added according to the present invention at a concentration of more than one mole per liter. Even more advantageously according to the present invention, a mixture of chaotropic agent and adsorption modifying agent is added to the reaction medium containing the cells of the pathogenic organisms.

 Advantageously according to the present invention, the step of concentrating DNA and / or RNA, which follows that of opening the cells, is carried out by reversible adsorption of the nucleic acids on a solid support containing an adsorbent material, followed by a elution of these acids.

 The DNA and / or RNA concentration step according to the present invention corresponds to an extraction and a purification of DNA and / or RNA.

 According to the present invention, the adsorbent material is advantageously a glassy material, preferably comprising at the surface hydroxy groups, or a silica-based compound.

 According to the present invention, the elution of the nucleic acids is advantageously carried out by washing the adsorbent material using at least one solvent containing monovalent ions, advantageously chosen from the group consisting of sodium thiocyanate and EDTA. The solvent according to the invention is thus advantageously a complexing agent formed from monovalent ions.

 The Applicant has thus surprisingly discovered that the elution of nucleic acids from the adsorption support could not be carried out without first converting the complex of nucleic acids and bivalent ions, formed during the first stage of concentration of organisms, in a complex consisting of nucleic acids and monovalent ions. Indeed, the complex of nucleic acids and bivalent ions is strongly linked to the solid adsorption support and cannot be desorbed and then eluted by simple washing using a solvent. Desorption, followed by elution, of nucleic acids is only made possible by the addition of a complexing agent formed

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 of monovalent ions. Such an agent thus makes it possible to exchange the bivalent ions, linked to the nucleic acids, for monovalent ions and thus to form a new complex which will be able to be desorbed much more easily than the complex nucleic acids / bivalent ions. After completion of the ion exchange, a solvent is then added to finalize the elution of the nucleic acids. This solvent must be compatible with the amplification step which follows the nucleic acid concentration step, and is advantageously chosen from the group consisting of water or a diluted buffer.

 Advantageously according to the present invention, the method can also contain a volume concentration step, in order to achieve volumes of the order of a hundred u. I up to less than 1 ml, advantageously less than 100 μl, before proceeding with the amplification of the nucleic acids and following the step of concentrating the nucleic acids.

 Advantageously according to the present invention, the step of detecting pathogenic organisms, which follows that of amplifying the nucleic acids, is an online detection (in real time), in situ. Even more advantageously, this detection is carried out by fluorescence, by luminescence or by mass spectrometry. It can also be carried out by other detection techniques well known to those skilled in the art, such as those of hybridization on a chip or of enzymatic amplification.

 Even more advantageously according to the invention, the detection is carried out in situ by fluorescence. In order to obtain a detection signal, it is thus possible to add, during the amplification step, Cybergreen or another intercalating fluorophore with two strands making it possible to indicate the success of the amplification by a strong increase in fluorescence at course of the cycle. In fact, the primers labeled with fluorophores specifically hybridize with the target DNA of the sample.

As the PCR amplification reaction generates a large number of copies of the target DNA, it is thus possible to obtain a quantification of the intensity of the fluorescence signal, directly correlated with the quantity of initial target DNA in the sample analyzed.

 To obtain multispecific detection, i.e. specific detection of several pathogens, in a single bottle and in order to increase the specificity vis-à-vis these pathogens if necessary

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 Molecular or similar tracers can be added to the different vials used during amplification. Using standard instrumentation, up to four different species can be detected per bottle.

 Advantageously, the detection method according to the present invention may also contain a prior filtration step, before proceeding to the step of concentrating the pathogenic organisms. Such filtration can be useful in the case of the analysis of raw water or surface water samples, particularly contaminated and loaded with troublesome substances such as sand.

 Advantageously, the detection method according to the present invention may also contain a preliminary step of treatment of the water sample by thermal shock and / or by adding a gene-inducing chemical compound in order to induce the RNA of specific genes and detect viable pathogenic organisms in said water sample. Indeed, a thermal shock (increase in temperature for a few moments) or the addition of a chemical compound induce a set of proteins allowing the realization of an RT reaction (reverse transcriptase) as a first step, when the first PCR is carried out, thus making it possible to obtain a specific signal from viable organisms. The RT reaction transforms RNA into DNA. If necessary, DNAases can be added to destroy the native DNA. Specificity is obtained by inducing a specific high level DNA.

 Advantageously according to the present invention, a thermal shock is produced by heat pulse lasting less than 3 hours, advantageously at a temperature of 30 to 50 C.

hscB, hsIJ, hslV, htpG, htpX, htrB, htrC, pphA, pphB, rpoE et d'autres protéines de Advantageously according to the present invention, a gene-inducing chemical compound is added. Such a compound makes it possible to induce the RNA of specific genes. The compound is advantageously a nutritive element such as lactose or a targeted inducer such as IPTG (Isopropyl- (beta) -D-thiogalactopyranoside). This chemical compound allows a sharp increase in the level of specific RNA in cells. The duration of the induction depends on the organism, but is typically between 2 minutes and 1 hour. Heat induced proteins like dnaJ, grpE,

hscB, hsIJ, hslV, htpG, htpX, htrB, htrC, pphA, pphB, rpoE and other proteins from

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 shock (eg classes of small HSP, HSP 60, HSP 70, HSP 90, HSP 100, ubiquitin) are effective targets.

 In the context of the present invention, the induction of RNA of specific genes can also be carried out by UV irradiation, by the use of toxic substances such as cis-platinum, anisomycin, tunicamycin, arsenite or by osmotic shock.

 Induction is direct proof of the viability of the cell. In addition, an RNA can thus be detected in numerous copies with greater sensitivity compared to DNA. While DNA detection is important, since DNA is present in each cell in a fixed amount, and it is then easy to correlate DNA with the number of cells, RNA is it can be induced at different levels, thus making it possible to detect the viability of pathogenic organisms.

 Advantageously according to the present invention, the gene-inducing chemical compound is lactose or IPTG in order to induce the RNA of lac Z. The lac Z operon, which is unique in its sequence in a certain number of pathogenic organisms , is indeed a useful target. The duration of the induction is a few minutes.

 Advantageously according to the present invention, the detection of viable pathogenic organisms in a water sample is carried out after having obtained a first DNA detection signal representative of the presence of pathogenic organisms in said water sample. Thus, in a particular embodiment of the present invention, all of the steps of the method for detecting the presence of pathogenic organisms are first implemented in order to conclude on the actual (or not) contamination of the water sample analyzed. In the event of a positive response, by obtaining a DNA signal representative of the presence of pathogenic organisms in said sample, all of the steps of the detection method according to the present invention are again implemented, in adding a preliminary step of treatment of the water sample by thermal shock and / or by adding a gene-inducing chemical compound in order to induce the RNA of specific genes. The DNA signal is then compared with the RNA signal obtained, which makes it possible to detect viable pathogenic organisms in the sample of water analyzed.

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 Advantageously according to the present invention, the control logic consists of at least one computer, for example of the PC type, which controls all of the steps. The control logic thus makes it possible to trigger and control the various stages of the process and to collect, at the end of the process, the analysis data relating to the detection of organisms.

 Advantageously according to the present invention, the analysis data relating to the detection of the organisms are automatically transferred to a central control system. The central control system according to the present invention advantageously consists of at least one computer. This central control system will be able to group, analyze and manage all the analytical data, for example using databases, and incorporate this data into mathematical models which will make it possible to predict the rate of contamination of networks d water analyzed, the type of microorganisms present in these networks and the survival rate of these microorganisms.

 Advantageously according to the present invention, the process operates continuously, autonomously, for a duration greater than or equal to one day, preferably from three days to seven days. The operating time of the process can vary depending in particular on the number of reagents used.

 In addition, a given water sample can be assayed and analyzed several times a day using the detection method according to the present invention. To do this, specific DNA sequences (primers) are used, and if necessary new primers can be developed. The duration of the assay for a test cycle using the method according to the present invention is optimized to a duration of the order of the doubling time of most organisms (2 to 6 hours).

 The present invention also relates to a device for detecting the presence of pathogenic organisms in a water sample, comprising: a unit for concentrating pathogenic organisms (1), a unit for opening cells for pathogenic organisms (2 ), a DNA and / or RNA concentration unit (3), a nucleic acid amplification unit (4),

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 a qualitative and / or quantitative detection unit of DNA and / or RNA (5) representative of the contamination of water by said pathogenic organisms; characterized in that it further comprises: at least one control logic (6) which manages the sequential processing steps implemented by the units (1) to (5) fully automatically, pumping means (7 ), automatic withdrawal and distribution means (8), and a central control system (9).

 Advantageously according to the present invention, the nucleic acid amplification unit (4) consists of at least one chain reaction apparatus by thermocycle polymerase. The thennocycle PCR apparatus according to the present invention performs successive nucleic acid amplification cycles, each cycle being carried out at a given temperature range and for a given duration. Even more advantageously according to the present invention, the amplification unit consists of two thermocycle polymerase chain reaction apparatuses in order to obtain maximum sensitivity with respect to the pathogens which it is desired to detect in water, especially when carrying out a "nested" PCR. The two devices are then used sequentially. The passage of nucleic acids from one device to another is carried out using automatic sampling and distribution means (8) and a control logic (6) which controls these means.

 In a particular embodiment of the present invention, a thermocycle apparatus with integrated online detection is used to detect amplification of specific DNA. This type of device thus consists of a thennocycle PCR device with integrated online detection chamber. In the particular case according to the present invention of carrying out an amplification by "nested" PCR, the second PCR is carried out in this type of apparatus, the first PCR being carried out in a conventional thermocycle apparatus. Such a device can be acquired from certain companies such as Roche Molecular Biochemicals. In this particular embodiment of the present invention, the amplification and detection units (4) and (5) are combined in the same device.

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 Advantageously according to the present invention, the concentration unit for pathogenic organisms (1) is a metal reactor, comprising a system for introducing the water sample, advantageously consisting of valves, a system for introducing different reagents. , advantageously made up of tubes, valves and rotary or roller pumps, a stirring system, a system for removing the supernatant liquid, advantageously made up of tubes and valves, a system for removing the precipitate, advantageously made up of valves and pumps, and advantageously placed at the bottom of the reactor, and a heating system, advantageously consisting of a heating jacket (sheath) placed around the reactor.

 The reactor according to the present invention is made of metal, advantageously of stainless steel, in order to obtain a high resistance to the chemical reagents introduced into said reactor. The reactor advantageously occupies a volume of 100 ml to 10 liters, preferably from 1 to 3 liters. The tubes are advantageously made of steel or plastic.

 The introduction of the water sample according to the present invention is carried out automatically by means of valves which are opened and closed using the control unit (6). According to the present invention, the introduction of the various reagents necessary for the concentration of pathogenic organisms is carried out using tubes, valves and rotary or roller pumps, said valves and pumps also being actuated by the control logic ( 6). Agitation and heating are also controlled by the control unit (6). The internal temperature of the reactor is advantageously controlled using sensors.

 Advantageously according to the present invention, the cell opening unit (2) contains a pearl mill. The beads used are advantageously chemically inert and must be strong enough to cause the cells to rupture. The beads used according to the present invention are advantageously small beads, such as steel or polymer beads, such as plastic, the diameter of which can vary from a few mm to a few tens of µm. In order to improve the cracking of the cells of pathogenic organisms, the beads are agitated by a strong source of energy. The possible sources of energy for shaking the pearls can be

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 sources of ultrasound, agitators of the rotary agitator type, Ultradurax type devices, sonotrode type devices, or strong vibrators.

 Advantageously according to the present invention, the DNA and / or RNA concentration unit (3) contains one or more solid support (s), containing a reversible adsorption material for nucleic acids, advantageously a material glassy. The concentration of nucleic acids can thus be carried out for example using a porous absorption column containing a glassy material, advantageously comprising hydroxy groups on the surface, or a silica-based material, which will allow the reversible adsorption of nucleic acids on the solid support.

 Even more advantageously according to the present invention, the solid supports are constituted by micro-titration plates containing multi-wells, advantageously plates of 60.96 or 384 wells, which themselves contain adsorbent materials. Each well then corresponds to a reversible adsorption micro-column.

 Even more advantageously according to the present invention, the wells of the micro-titration plates are used only once and are moved automatically to receive the liquid sample coming from the opening unit of the cells of the organisms. Pressurized air can be injected to drive the fluid into the wells. The plate is referenced with respect to an XY coordinate system, X corresponding to the axis along the width of the plate and Y corresponding to the axis along the length of the plate. The automatic displacement of the plate, and consequently that of the wells which are fixed on the plate, is carried out along the X and Y axes, for example using the arm of a robot. The automatic movement can be actuated by the control unit according to the present invention. In a particular embodiment of the present invention, the automatic movement is carried out using a robot, which is controlled using a control logic (6), advantageously constituted by one or more computers ( s). Advantageously, the robot has an arm which can move in space, according to an XYZ frame of reference.

 Advantageously according to the present invention, the wells of the microtitration plates can be protected against evaporation by a drop of fluid having a low specific density or by a transparent cover.

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 After elution of the different nucleic acid samples present in the wells used during the adsorption, all the liquid samples are collected in order to be able to carry out the following steps of amplification of the nucleic acids and detection of pathogenic organisms.

 Advantageously according to the present invention, the detection unit (5) is constituted by a fluorescence detector. The fluorescence detector advantageously comprises an optical module such as an electronic camera which records the photons from the fluorophores and which converts them into electrons. The electronic image formed is then transferred to the control unit (6).

 The operation of the device according to the invention will be better understood on reading the description given below with reference to the appended FIG. 1 which schematically illustrates a particular embodiment of the present invention.

 The detection device according to the present invention comprises at least one unit for concentrating pathogenic organisms (1), one unit for opening cells of pathogenic organisms (2), one unit for concentrating (adsorption then elution) of nucleic acids (3 ), a nucleic acid amplification unit (4), a nucleic acid detection unit (5), a control logic (6) constituted by a PC-type computer which controls the entire device, means of pumping (7), a means of automatic withdrawal and distribution (8) constituted by a robot, a central control system (9), constituted by at least one computer, in interface with a network (13) which makes it possible to transfer the data from the control unit (6) to the central control system (9), a cooling zone (10), a water sample inlet and outlet system (11) and an area (12) to store the reagents necessary for the units (1 ), (2), (3), (4) and (5).

 Advantageously according to the present invention, the sample containing the pathogenic organisms circulates from the pathogenic organism concentration unit (1) to the entry of the nucleic acid amplification unit (4), via the units d opening the cells (2) and concentrating the nucleic acids (3), using pumping means (7), advantageously constituted by tubes fitted with valves and / or pumps. The pumping means (7) thus constitute a fluidic system

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 allowing to connect the units (1), (2), (3) and (4). The pumping means (7) according to the invention are actuated and controlled by the control logic (6).

 Advantageously according to the present invention, the manipulation of the nucleic acids, after completion of the DNA and / or RNA concentration, is carried out from the inlet of the nucleic acid amplification unit (4) up to at the outlet of the nucleic acid detection unit (5), using automatic sampling and distribution means (8), advantageously constituted by a robot. Thus, the units (4) and (5) do not have a direct connection between them (no fluidic system), unlike the units (1) to (4). The automatic withdrawal and distribution means (8) according to the invention are actuated and controlled by the control logic (6).

 Advantageously according to the present invention, the control logic (6) consists of at least one computer, for example of the PC type, which controls the entire device and which receives, manages and processes all of the relative analysis data. the detection of pathogenic organisms.

 Advantageously, the control logic (6) according to the present invention is interfaced with a network for transferring, automatically or on request, the analysis data relating to the detection of organisms to a central control system (9).

 Advantageously according to the present invention, the central control system (9) consists of at least one computer. The central control system (9) according to the present invention can thus receive and record the acquired data, which are then analyzed, checked and managed.

 Advantageously according to the present invention, the device further contains a cooling zone (10) for storing the sensitive reagents, preferably a cooling block, which is protected against condensation of water vapor.

 Advantageously according to the present invention, the device is directly connected to the drinking water distribution network for automatic sampling. The detection device according to the present invention thus makes it possible to test in a fully automated manner samples taken from different water networks, in particular that of drinking water. It can also be reused many times, without the need to change the different units used.

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 Typically, the succession of the different stages of the detection method according to the present invention can be advantageously implemented as follows: 1. The unit (1) for the concentration of pathogenic organisms is purged, cleaned and then filled with a volume of 100 ml 5 liters of water, preferably 2 to 3 liters of water. Water is introduced into the reactor using valves, the opening of which is actuated by a computer (6).

2. Various reagents, such as sodium carbonate and calcium acetate, are added to the reactor via valves and rotary pumps, which are controlled by the computer (6). An inclusion precipitate is then formed at the bottom of the chamber, on which cells and nucleic acids of pathogenic organisms are absorbed.

3. The supernatant is removed, for example by gravity, using a tube and a valve.

4. The precipitate is dissolved using an acid, added to the reactor via a pump, which is activated by the computer (6). A buffering agent is also added in the same manner.

5. The solution containing the pathogenic organisms is transferred to the unit (2) for opening the cells of the organisms, constituted by a pearl mill, by gravity or by the action of a pump controlled by the 'computer (6). At this stage, the volume of solution entering the pearl mill is 10 to 100 ml, preferably 50 ml.

6. The cells are opened by grinding on agitated pearls, by means of mechanical energy, for example with a rotary or vibrating agitator or a source of ultrasound.

7. The ruptured cells are then transferred, via a computer-controlled pump (6), to a plastic container of approximately 100 ml.

8. A chaotropic agent and an adsorption modifying agent are added via valves and pumps, controlled by the computer (6).

9. The DNA and / or RNA of the cells is then adsorbed on a solid support, advantageously on micro-titration plates containing wells, said wells

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 themselves containing an adsorbent material, such as a vitreous material. Pressurized air can be injected to drive the fluid into the wells and an XY or XYZ repository is used to manipulate the matrix of adsorbent columns. The manipulation of the wells, which are each of the micro-columns, can thus be carried out using a robot actuated by the computer (6), the arm of which moves the plate where the wells are located according to an XY reference system. or XYZ.

 10. A monovalent complexing agent is then added via valves and pumps, controlled by the computer (6), in order to replace the bivalent ions complexed with nucleic acids by monovalent ions. The nucleic acid / monovalent ion complex thus formed is then at this stage always linked to the adsorption microcolumns.

 11. The nucleic acids are then eluted by adding a buffer, such as TRIS / EDTA or water, using rotary pumps.

12. The nucleic acids are collected in a tube manipulated for example by a robot. From this stage, all the manipulations are made using an automatic sampling and distribution means such as a robot (8).

13. Optionally, and if necessary, the total volume of solution containing the different nucleic acids collected and eluted is reduced to reach volumes of the order of a hundred ni or less than 1 ml, before amplifying the acids.

14. The nucleic acids are transferred to a standard thermocycle device, using the robot (8) and the first amplification is then carried out in the device by first adding the reagents to carry out the PCR such as the PCR buffer, primer set 1, TAQ, nucleotides.

15. The mixture of acids amplified for the first time is then separated into several vials in order to obtain maximum sensitivity vis-à-vis the pathogens that one seeks to detect.

16. A second amplification is then carried out in a second real-time thermocycle apparatus, in particular by adding the set of primers 2 (TAQ, nucleotides). Visualization and detection agents, such as fluorescent agents, are also added. Detection agents will thus be able to hybridize on the fragments of the amplified germs. All sample handling (transfers,

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 additions of the different reagents, ...) are handled using the robot (8) mentioned above.

17. The fluorescence is checked and detected in situ during the second amplification (online detection).

18. The analysis data relating to the detection of pathogenic organisms are displayed on the computer (6) and transferred to a central control system (9). All the sequential steps of the process are managed and controlled by a control logic (6) constituted by a computer.

 The following examples are given without limitation and illustrate the present invention.

Examples of embodiment of the invention: Example 1: Precipitation by inclusions of pathogenic agents A water sample is first collected, taken directly from the drinking water supply network, from the environment or from laboratory or other source, in a 2 liter reactor and heated to 50 C.

10 ml of TRIS 1 M (buffering agent) are first added to reach a pH of 8.5 and 25 ml of 1M calcium acetate are then added. The two compounds are then mixed quickly, then the mixing operation is stopped.

10 ml of 1 M Na2CO3 are then added and the various compounds are reacted for 3 minutes. Mix carefully with about 5 seconds, then add 10 ml of Na2CO3 and then react the reaction medium for another 3 minutes. Gently but sufficiently mixed and the reaction medium is again reacted for another 10 minutes. 7.5 ml of IM calcium acetate are added and the reaction is carried out for 3 minutes. Mix carefully, add 10 ml of Na2CO3 and react further for 5 minutes.

The supernatant is decanted by opening a valve. The precipitate is dissolved by adding 13 ml of 20% formic acid. The mixture is then buffered by adding 2 M TRIS base to raise the pH to 7.2.

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Example 2: PCR Amplification and Detection 2.1 A thermocycle apparatus with integrated online detection is used to detect the amplification of the specific DNA, such as the LightCycler apparatus marketed by the company Roche Molecular Biochemicals. 32 samples are handled in parallel, and 40 amplification cycles are carried out at high temperature (from 75 C to 100 C), the 40 cycles lasting approximately 45 minutes. Amplification and detection take place in capillaries. The detection threshold is around 1 pathogenic organism.

2.2 As an alternative, it is also possible to use the ABI prism 7700 detection system, sold by the company PE Biosystems, processing 96 samples in parallel (two hours), during 40 cycles for the detection of more than 10 samples in a vial, lasting approximately 2.5 hours.

 Sensitivities of the order of a few femtograms of total DNA / RNA are thus obtained (which is equivalent to an isolated cell). The detection threshold is around 1 pathogenic organism.

Claims (35)

1. Method for detecting the presence of pathogenic organisms in a water sample comprising the following sequence of steps: a concentration of pathogenic organisms, an opening of the cells of pathogenic organisms, a concentration of DNA and / or RNA, at least one amplification of nucleic acids, and a qualitative and / or quantitative detection of DNA and / or RNA representative of the contamination of water by said pathogenic organisms; characterized in that all of the sequential steps of the process are managed entirely automatically using a control logic.  2. Detection method according to claim 1, characterized in that the pathogenic organisms detected are bacteria, viruses, fungi and / or protozoa.  3. Detection method according to claim 1 or 2, characterized in that the initial volume of water sample is greater than 50 ml.  4. Detection method according to any one of claims 1 to 3, characterized in that the amplification of nucleic acids is carried out by a polymerase chain reaction in real time.  5. Detection method according to any one of claims 1 to 4, characterized in that the concentration of pathogenic organisms is carried out by precipitation, co-precipitation or precipitation by inclusion.  6. Detection method according to claim 5, characterized in that the concentration is carried out in the presence of carbonates of bivalent ions, advantageously chosen from the group consisting of calcium carbonate, strontium carbonate and barium carbonate.  7. Detection method according to any one of claims 1 to 6, characterized in that the opening of the cells is carried out by grinding on agitated pearls. <Desc / Clms Page number 25>  8. Detection method according to claim 7, characterized in that the pearls are agitated by ultrasound, by mechanical agitation or by mechanical vibration.  9. Detection method according to any one of the preceding claims, characterized in that, during the opening or following the opening of the cells and prior to the concentration of DNA and / or RNA, are added to the reaction medium containing the cells at least one chaotropic agent, advantageously a guanidine salt such as guanidine thiocyanate, and at least one adsorption modifying agent, advantageously an alcohol such as ethanol.  10. Detection method according to any one of the preceding claims, characterized in that the concentration of DNA and / or RNA is carried out by reversible adsorption of the nucleic acids on a solid support containing an adsorbent material, followed by elution of these acids.  11. Detection method according to claim 10, characterized in that the elution of the nucleic acids is carried out by washing the adsorbent material using at least one solvent containing monovalent ions, advantageously chosen from the group consisting of sodium thiocyanate and EDTA.  12. Detection method according to any one of the preceding claims, characterized in that the detection of pathogenic organisms is an online detection, advantageously carried out by fluorescence, by luminescence or by mass spectrometry.  13. Detection method according to any one of the preceding claims, characterized in that it further comprises a prior filtration step.  14. Detection method according to any one of the preceding claims, characterized in that it further comprises a preliminary step of treatment of the water sample by thermal shock and / or by addition of a chemical compound inducing gene in order to induce the RNA of specific genes and to detect viable pathogenic organisms in said water sample.  15. Detection method according to claim 14, characterized in that the thermal shock is produced by a heat pulse lasting less than 3 hours.  16. Detection method according to claim 14 or 15, characterized in that the gene-inducing chemical compound is lactose or IPTG in order to induce the RNA of lake Z. <Desc / Clms Page number 26>  17. Detection method according to any one of the preceding claims, characterized in that the control logic consists of at least one computer.  18. Detection method according to any one of the preceding claims, characterized in that the analysis data relating to the detection of the organisms are automatically transferred to a central control system.  19. Detection method according to any one of the preceding claims, characterized in that the method operates continuously, autonomously, for a duration greater than or equal to one day, preferably from three days to seven days.  20. Device for detecting the presence of pathogenic organisms in a water sample, comprising: a unit for concentrating pathogenic organisms (1), a unit for opening cells for pathogenic organisms (2), a unit for concentrating of DNA and / or RNA (3), a nucleic acid amplification unit (4), a qualitative and / or quantitative detection unit of DNA and / or RNA (5) representative of the contamination of water by said pathogenic organisms; characterized in that it further comprises: at least one control logic (6) which manages the sequential processing steps implemented by the units (1) to (5) fully automatically, pumping means (7 ), automatic withdrawal and distribution means (8), and a central control system (9).  21. Detection device according to claim 20, characterized in that the nucleic acid amplification unit (4) consists of at least one thermocycle polymerase chain reaction device.  22. Detection device according to claim 20 or 21, characterized in that the pathogen concentration unit (1) is a metal reactor comprising a system for introducing the water sample, a system for introduction of different reagents, a stirring system, an evacuation system <Desc / Clms Page number 27>  supernatant, a system for removing the precipitate, advantageously placed at the bottom of the reactor, and a heating system, advantageously consisting of a heating jacket placed around the reactor.  23. Detection device according to any one of claims 20 to 22, characterized in that the cell opening unit (2) contains a pearl mill.  24. Detection device according to any one of claims 20 to 23, characterized in that the DNA and / or RNA concentration unit (3) comprises one or more solid support (s) containing a reversible adsorption material for nucleic acids, advantageously a glassy material.  25. Detection device according to claim 24, characterized in that the solid supports consist of micro-titration plates containing multi-wells, advantageously plates of 60.96 or 384 wells.  26. Detection device according to claim 25, characterized in that the wells of the micro-titration plates are used only once and are moved automatically to receive the liquid sample coming from the cell opening unit organisms.  27. Detection device according to claim 26, characterized in that the automatic displacement of the wells is carried out using a robot.  28. Detection device according to any one of claims 20 to 27, characterized in that the detection unit (5) consists of a fluorescence detector, advantageously comprising an electronic camera.  29. Detection device according to any one of claims 20 to 28, characterized in that the sample containing the pathogenic organisms circulates from the pathogen concentration unit (1) to the entrance to the unit amplification of nucleic acids (4), via cell opening units (2) and

 nucleic acid concentration (3), using pumping means (7), advantageously constituted by tubes fitted with valves and / or pumps.  30. Detection device according to any one of claims 20 to 29, characterized in that the manipulation of the nucleic acids, after achievement of the DNA and / or RNA concentration, is carried out from the input of the nucleic acid amplification unit (4) until it leaves the detection unit for <Desc / Clms Page number 28>  nucleic acids (5), using automatic sampling and distribution means (8), advantageously constituted by a robot.  31. Detection device according to any one of claims 20 to 30, characterized in that the control logic (6) consists of at least one PC-type computer which controls the entire device.  32. Detection device according to any one of claims 20 to 31, characterized in that the control logic (6) is interfaced with a network for transferring, automatically or on request, the analysis data relating to detection of organisms at a central control system (9).  33. Detection device according to any one of claims 20 to 32, characterized in that the central control system (9) consists of at least one computer.  34. Detection device according to any one of claims 20 to 33, characterized in that the device also contains a cooling zone (10) for storing the sensitive reagents, preferably a cooling block.  35. Detection device according to any one of claims 20 to 34, characterized in that the device is directly connected to the drinking water distribution network for automatic sampling.