Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa

Definitions

• the present invention relates to the field of microbiological diagnosis, detection techniques allowing the identification and the quantification of microorganisms present in fluids and products such as for example water.
• microbiological identification methods require a culture step on selective media followed in general by identification, according to morphological, biochemical, and / or immunological characteristics.
• faecal contamination indicators do not, however, make it possible to predict the presence of bacterial contamination of non-faecal origin (Pseudomonas aeruginosa, Legionella ”) as well as non-bacterial contamination (viruses and parasites).
• EP-A-0 453 290 and US-A-5,558,989 describe a method for detecting the pathogenic species in humans, Giardia lamblia, based on the use of nucleic probes (DNA and / or RNA) corresponding to the sequence of 18S rRNA.
• EP-A-0 550 883 describes a PCR test with reagents to search for G. lamblia, the sensitivity of which is 1-5 oocysts / ml of water concentrate. Molecular methods distinguishing dead parasites from viable and / or infectious parasites thus making it possible to better appreciate the real health risk posed by the presence of these parasites in water have been described.
• WO-A-97/42349 which relates to the detection of viable Cryptosporidium and Giardia (by detection of mRNA of thermal shock proteins hsp 70) and / or infectious (cell culture and enzymatic amplification)
• US-A-5,556,774 which relates to a method for detecting Cryptosporidium viable by combination of a PCR step and an in vitro excystation step.
• FISH in situ hybridization technique
• a nucleotide fragment, or an oligonucleotide, or a polynucleotide is a chain of nucleotide motifs assembled together by phosphoric ester bonds, characterized by the informational sequence of natural nucleic acids, capable of hybridizing to a nucleotide fragment under conditions predetermined, the sequence being able to contain monomers of different structures and to be obtained from a natural nucleic acid molecule and / or by genetic recombination and / or by chemical synthesis.
• a nucleotide motif is derived from a monomer which may be a natural nucleotide of nucleic acid, the constituent elements of which are a sugar, a phosphate group and a nitrogenous base; in DNA the sugar is deoxy-2-ribose, in RNA the sugar is ribose; depending on whether it is DNA OR RNA, the nitrogen base is chosen from adenine, guanine, uracil, cytosine, thymine; or else the monomer is a nucleotide modified in at least one of the three aforementioned constituent elements; by way of example, the modification can take place either at the base level, with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6- purine, bromo-5-deoxyuridine or any other modified base capable of hybridization, either at the sugar level, for example the replacement of at least one deoxyribose
• sequence is meant any ordered sequence of nucleotide type patterns, the chemical nature and order in a reference sense constitute information of the same quality as that of natural nucleic acids.
• hybridization is meant the process during which, under appropriate conditions, two nucleotide fragments, having sufficiently complementary sequences are capable of forming a double strand with stable and specific hydrogen bonds.
• a nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner.
• the hybridization conditions are determined by the stringency, that is to say the rigor of the operating conditions. Hybridization is all the more specific as it is performed at higher stringency. Stringency is defined in particular as a function of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids.
• the stringency can also be a function of the parameters of the reaction, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and / or the hybridization temperature.
• the stringency of the conditions under which a hybridization reaction must be carried out will depend mainly on the probes used. All these data are well known and the appropriate conditions can be determined by a person skilled in the art. In general, depending on the length of the probes used, the temperature for the hybridization reaction is between about 20 and 65 ° C, in particular between 35 and 65 ° C in saline at a concentration of about 0.8 to 1 molar.
• a probe is a nucleotide fragment comprising from 5 to 100 monomers, in particular from 6 to 35 monomers, having a specificity of hybridization under determined conditions to form a hybridization complex with a nucleotide fragment having, for example, a nucleotide sequence included in a ribosomal RNA, the DNA obtained by reverse transcription of said ribosomal RNA and the DNA (here called ribosomal DNA or rDNA) of which said ribosomal RNA is the transcription product; a probe can be used for diagnostic purposes (in particular capture or detection probes).
• a capture probe is immobilized or immobilizable on a solid support by any appropriate means, that is to say directly or indirectly, for example by covalence or adsorption.
• a detection probe can be labeled using a marker chosen from radioactive isotopes, enzymes (in particular a peroxidase, an alkaline phosphatase, or an enzyme capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate), chemical compounds chromophores, chromogenic compounds, fluorigenes or luminescent, analogs of nucleotide bases, and ligands such as biotin.
• a marker chosen from radioactive isotopes, enzymes (in particular a peroxidase, an alkaline phosphatase, or an enzyme capable of hydrolyzing a chromogenic, fluorigenic or luminescent substrate), chemical compounds chromophores, chromogenic compounds, fluorigenes or luminescent, analogs of nucleotide bases, and ligands such as biotin.
• a primer is a probe comprising from 5 to 100, preferably from 10 to 40 nucleotide units and having a specificity of hybridization under conditions determined for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), in a sequencing process, in a reverse transcription method, etc.
• PCR Polymerase Chain Reaction
• the probes and primers according to the invention are chosen from:
• identification sequence we mean any sequence or any fragment as defined above, which can serve as a detection and / or capture probe.
• treatment of the aqueous medium is meant any filtration and / or lysis and / or purification step.
• lysis step we mean a step capable of releasing the nucleic acids contained in the protein and / or lipid envelopes of microorganisms (such as cellular debris which disturb subsequent reactions).
• the lysis methods as described in the applicant's patent applications can be used:
• WO-A-99/15321 on mechanical lysis.
• Those skilled in the art will be able to use other well-known lysis methods such as thermal or osmotic shocks or chemical lyses with chaotropic agents such as guanidium salts (US-A-5,234,809).
• - By purification step is meant the separation between the nucleic acids of the microorganisms and the cellular constituents released in the lysis step. This step generally makes it possible to concentrate the nucleic acids.
• the intermediate layer is itself covered by an external layer based on a polymer capable of interacting with at least one biological molecule, the external polymer is thermosensitive and has a predetermined lower critical solubility temperature (LCST) of between 10 and 100 ° C and preferably between 20 and 60 ° C.
• LCST lower critical solubility temperature
• This outer layer is synthesized from cationic monomers, which generate a polymer with the ability to bind nucleic acids.
• This intermediate layer isolates the magnetic charges from the nucleus, in order to avoid the problems of inhibiting the techniques of amplification of these nucleic acids.
• nucleic acid purification method is the use of silica either in the form of a column (Qiagen kits for example) or in the form of inert particles [Boom R. et al., J. Clin. Microbiol., 1990, n ° 28 (3), p. 495-503] or magnetic (Merck: MagPrep ® Silica, Promega: MagneSil TM Paramagnetic particles).
• Other widely used methods are based on ion exchange resins in columns (Qiagen kits for example) or in paramagnetic particle format (Whatman: DEAE-Magarose) [Levison PR et al., J. Chromatography, 1998, p. 337-344].
• Another very relevant method for the invention is that of adsorption on a metal oxide support (Xtrana company: Xtra-Bind TM matrix).
• detection step is meant either direct detection by a physical method, or a detection method using a marker.
• a hybridization method using specific probes is implemented for the detection step.
• This particular embodiment consists in bringing the nucleic acids, amplified or not, into contact with the microorganisms to be detected with a capture probe fixed on a solid support, and capable of specifically hybridizing with said nucleic acids; then to reveal, according to known methods, the possible presence of the nucleic acids attached to the solid support, in particular by means of at least one capture probe.
• marker is meant a tracer capable of generating a signal.
• a non-exhaustive list of these tracers includes the enzymes which produce a detectable signal for example by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-lactactosidase, glucose-6-phosphate dehydrogenase; chromophores such as fluorescent, luminescent or coloring compounds; groups with electron density detectable by electron microscopy or by their electrical properties such as conductivity, by amperometry or voltametry methods, or by impedance measurements; the groups detectable by optical methods such as diffraction, surface plasmon resonance, variation of contact angle or by physical methods such as atomic force spectroscopy, tunnel effect, etc.
• the polynucleotide can be labeled during the enzymatic amplification step, for example using a labeled nucleotide triphosphate for the amplification reaction.
• the labeled nucleotide will be a deoxyribonucleotide in DNA generating amplification systems, such as PCR, or a ribonucleotide in RNA generating amplification techniques, such as TMA or NASBA techniques.
• the polynucleotide can also be labeled after the amplification step, for example by hybridizing a labeled probe according to the sandwich hybridization technique described in document WO-A-91/19812.
• Another particular preferred mode of labeling nucleic acids is described in Application FR-A-2,780,059 to the Applicant.
• Another preferred mode of detection uses the 5'-3 'exonuclease activity of a polymerase as described by Holland P.M., PNAS (1991) p 7276-7280.
• Signal amplification systems can be used as described in WO-A-95/08000 and, in this case, the preliminary enzymatic amplification reaction may not be necessary.
• enzymatic amplification is meant a process generating multiple copies of a particular nucleotide fragment using specific primers by the action of at least one enzyme.
• amplification of nucleic acids there are, among others, the following techniques: - PCR (Polymerase Chain Reaction), as described in patents US-A-4,683,195, US-A-4,683,202 and US-A- 4,800,159,
• solid support includes all materials on which a nucleic acid can be immobilized. Synthetic materials, or natural materials, optionally chemically modified, can be used as solid support, in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose, or dextran; polymers, copolymers, in particular based on styrene type monomers, natural fibers such as cotton, and synthetic fibers such as nylon; mineral materials such as silica, quartz, glasses, ceramics; latexes; magnetic particles; metal derivatives, gels etc.
• the solid support can be in the form of a microtiter plate, a membrane as described in application WO -A-94/12670, a particle or a biochip.
• biochip a solid support of reduced size where a multitude of capture probes are fixed at predetermined positions.
• the main characteristic of the solid support must be to retain the hybridization characteristics of the capture probes on the nucleic acids while generating minimum background noise for the detection method.
• An advantage of biochips is that they simplify the use of numerous capture probes thus allowing multiple detection of microorganisms to be detected while taking into account the polymorphism of said microorganisms to be detected
• the invention described below makes it possible to solve the problems posed by the methods described above, both in terms of sensitivity, specificity and multidetection capacity, while being quick and easy to implement.
• a first object of the invention is a method for controlling the microbiological quality of an environmental aqueous medium, capable of comprising different microorganisms, comprising the following steps: - a reference set is chosen, consisting of at least three microorganisms, representative, together or separately, of a level of microbiological quality,
• a microbiological determination kit consisting of at least three probes for specifically and respectively identifying said three microorganisms, - after treatment of the medium to be analyzed, we put - said microorganisms, or any fraction obtained from them, in contact with said determination kit, whereby we multi-determine said microorganisms, this determination being representative of the level of microbiological quality middle.
• the invention also relates to a microbiological determination kit comprising a mixture of bacteria and / or virus and / or parasite identification probes, said identification probes each being specific for a species or at least of a genus bacteria, viruses or parasites that may be present in a sample of liquid to be measured.
• a kit designates any manual, semi-automatic or automatic method allowing the implementation of a dosing means, dosing signifying the identification and / or determination of viability and / or quantification, each of these three parameters being determined in sequence or according to combinations: identification only; identification and quantification; identification and viability; identification, quantification and sustainability.
• This invention also relates to a multidetection method using in particular the biochip technique to search for a large number of microbiological parameters including contamination indicators required in the various legislations (USA, France, Europe) and pathogenic microorganisms including bacteria, viruses and parasites.
• a complete microbiological analysis of a sample can be carried out quickly, for example around 4 hours, and with great sensitivity, for example of the order of 1 micro target / 101-1001 thanks to step d 'enzymatic amplification.
• This multidetection method is specific to the species sought through the use of sequences, called identification sequences of each species, as probe, and can make it possible to determine the viability of microorganisms by the detection of viability markers such as, for example, rRNA and / or mRNA.
• this multidetection method makes it possible to apply it indifferently to any environmental aqueous medium, ie any aqueous medium to the exclusion of any bodily fluid.
• this method applies to all water intended for human consumption, clean industrial water, urban waste water and industrial, water for the food industry, process water and any fluid or product.
• microorganisms sought the following microorganisms will be cited by way of example: Among the bacteria: Escherichia coli, Escherichia coli SEROTYPE 0157: H7,
• Helicobacter pylori Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium perfringens, Staphylococcus epidermatitis, Staphylococcus aureus, Campylobacter coli, Campylobacter aeri, Campylobacter coli , Acinetobacter baumanii, Burkholderia gladioli, Burkholderia cepacia, Stenotrophomonas maltophilia, the genus Mycobacterium, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium simiae, Mycobacterium kansasii, Mycobacterium xenopi genus, Mycobacterium marinum
• the genus Cryptosporidium such as Cryptosporidium parvum
• the genus Giardia such as Giardia lamblia and Microsporidia.
• Microorganisms can be sought at the level of the genus to which they belong, either at the lower taxonomic level, ie at the level of the species, or at the level of serotypes, subtypes and in epidemiology: for example for Legionella, the determination be carried out by the identification sequence SEQ ID NO: 9 for research at the genus level and by SEQ ID NO: 10 or 11 for a determination by a specific identification sequence of the bacterium Legionella pneumophila.
• identification sequences corresponding to the species sought will be chosen from the sequences whose list is attached in the appendix of SEQ ID NO: 1 to SEQ ID NO: 104.
• the three identification probes which it comprises have at least one sequence chosen from any one of the sequences SEQ ID Nos: 1-104, and all fragments thereof comprising at least 5 contiguous monomers included in any one said sequences and the sequence of which has at least 70% identity with said any sequence.
• It comprises at least one identification probe specific for a bacterium, at least one identification probe specific for a parasite and at least one identification probe specific for a virus; preferably, it comprises at least one identification probe chosen from SEQ ID NO: 1 to SEQ ID NO: 39, SEQ ID NO: 62, SEQ ID NO: 61, SEQ ID NO: 66 to SEQ ID NO: 69 and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence; at least one identification probe chosen from SEQ ID NO: 40 to SEQ ID NO: 49, SEQ ID NO: 63 to SEQ ID NO: 65, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence; and at least one sequence chosen from among SEQ ID NO: 50 to SEQ ID NO: 60, and SEQ ID NO: 70 to SEQ ID NO: 104, and all fragments thereof comprising at least 5
• identification probes specific to at least four different bacteria include at least four identification probes specific to at least four different bacteria; preferably, they are chosen from SEQ ID NO: 1 to SEQ ID NO: 39, SEQ ID NO: 62, SEQ ID NO: 61, SEQ ID NO: 66 to SEQ ID NO: 69 and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• It comprises at least five identification probes specific to at least five different viruses; preferably, they are chosen from SEQ ID NO: 50 to SEQ ID NO: 60, and SEQ ID NO: 70 to SEQ ID NO: 104, and all fragments thereof comprising at least 5 contiguous monomers included in one any of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the microbiological determination kit comprises at least two identification probes specific to at least two parasites; preferably, it is chosen from SEQ ID NO: 40 to SEQ ID NO: 49 and SEQ ID NO: 63 to SEQ ID NO: 65, and all fragments thereof comprising at least 5 contiguous monomers included in any one said sequences and the sequence of which has at least 70% identity with said any sequence.
• the microbiological determination kit comprises at least one identification probe specific for a bacterium and at least one identification probe specific for at least one parasite.
• it comprises at least one identification probe chosen from SEQ ID NO: 1 to SEQ ID NO: 39, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 67 to SEQ ID NO: 69, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence
• Said microorganisms in the microbiological determination kit are chosen from the following bacteria: Escherichia coli, genus Salmonella, Staphylococcus aureus.
• at least one identification probe for the kit is chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO9: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 15 , SEQ ID NO: 23, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• Said microorganisms in the microbiological determination kit are chosen from the following bacteria: Escherichia coli, genus Salmonella, Staphylococcus aureus, Clostridium perfringens.
• at least one probe for identifying said kit is chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 15 , SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 29, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% d identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, genus Salmonella, Staphylococcus aureus, Genus Cryptosporidium.
• at least one probe of said kit is chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 40 to SEQ ID NO: 44, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from the following microorganisms: genus Salmonella, Staphylococcus aureus, Gardia lamblia, Cryptosporidium parvum.
• at least one probe of said kit is chosen from SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 46 to SEQ ID NO: 49, SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, and all fragments thereof including at at least 5 contiguous monomers included in any of said sequences and whose sequence has at least 70% identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, Enterovirus, Genus Cryptosporidium, Preferably, at least one probe for identifying said kit is chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 53 to SEQ ID NO: 55, SEQ ID NO: 70 to SEQ ID NO: 75, SEQ ID NO: 40 to SEQ ID NO : 44, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the said microorganisms for the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, Escherichia coli serotype 0157: H7, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium stus , Enterovirus: polio virus, coxsackie virus A and B, Echovirus, Genus Cryptosporidium, Cryptosporidium parvum, genus Giardia, Giardia lamblia.
• At least one probe for identifying the kit is chosen from SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 29, SEQ ID NO: 40 to SEQ ID NO: 49, SEQ ID NO: 53 to SEQ ID NO: 55, SEQ ID NO: 61 to SEQ ID NO: 75, and all fragments thereof comprising at least 5 contiguous monomers included in any one said sequences and the sequence of which has at least 70% identity with said any sequence.
• the said microorganisms for the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, Escherichia coli serotype 0157: H7, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium stus , Enterovirus: polio virus, coxsackie virus A and B, Echovirus, Genus Cryptosporidium, Cryptosporidium parvum, genus Giardia, Giardia lamblia, Genus Legionella, Legionella pneumophila, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Campylobacter coli, Campylobacter coli Hepatitis A, Calicivirus: Norwalk and Sapporo Virus, Adenovirus, Rotavirus
• At least one probe for identifying the kit is chosen from SEQ ID NO: 1 to SEQ ID NO: 4, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 14 to SEQ ID NO: 20 , SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 29, SEQ ID NO: 40 to SEQ ID NO: 51, SEQ ID NO: 53 to SEQ ID NO: 55, SEQ ID NO: 56 to SEQ ID NO: 104, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the said microorganisms for the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, Escherichia coli serotype 0157: H7, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium stus , Enterovirus: polio virus, coxsackie virus A and B, Echovirus, Genus Cryptosporidium, Cryptosporidium parvum, genus Giardia, Giardia lamblia, Genus Legionella, Legionella pneumophila, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Campylobacter coli, Campylobacter coli of hepatitis A, Calicivirus: Norwalk and Sapporo Virus, Adenovirus, Rot
• At least one probe for identifying the determination kit is chosen from SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 9 to SEQ ID NO: 55, SEQ ID NO: 56 to SEQ ID NO : 104, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from the following bacteria: Escherichia coli, Escherichia coli SEROTYPE 0157: H7, Helicobacter pylori, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equus , Staphylococcus aureus, Campylobacter coli, Campylobacter jejuni r _Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Pseudomonas aeruginosa, Vibrio cholerae, Acinetobacter baumanii, Burkholderia gladioli, Burkholderia cepacia, Stenotrophomonas maltophilia, Mycobacterium mycobaciumia mycobacterium
• Said microorganisms of the microbiological determination kit are chosen from the following viruses: Adenoviruses, such as Adeno virus 40, Adeno virus 41 bis; Astroviruses, HAstV-1-2; Enteroviruses, such as Poliovirus, Coxsackievirus, Echovirus, Rotaviruses, Caliciviruses, such as Norwalk virus, Sapporo virus, and Hepatitis viruses such as Hepatitis A virus.
• viruses such as Adeno virus 40, Adeno virus 41 bis
• Astroviruses such as Poliovirus, Coxsackievirus, Echovirus, Rotaviruses, Caliciviruses, such as Norwalk virus, Sapporo virus
• Hepatitis viruses such as Hepatitis A virus.
• Said microorganisms in the microbiological determination kit are chosen from the following parasites:
• Said microorganisms of the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, Escherichia coli serotype 0157: H7, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis,
• Streptococcus equinus Clostridium perfringens, Genus Salmonella, Staphylococcus aureus, Enterovirus: polio virus, coxsackie virus A and B, Echovirus, Genus Cryptosporidium, Cryptosporidium parvum, genus
• At least one probe for identifying the necessary is chosen from SEQ ID NO: 1 to SEQ ID NO:
• SEQ ID NO: 97 and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and of which the sequence has at least 70% identity with any said sequence.
• Said microorganisms of the microbiological determination kit are chosen from the following microorganisms: Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium perfringens, Genus Salmonella, Staphylococcus aureus , coxsackie virus A and B, Echovirus, Genus Cryptosporidium, Cryptosporidium parvum, genus Giardia, Giardia lamblia, Genus Legionella, Legionella pneumophila, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Campylobacter coli, Campylobacter jejuni, Hepatitis virus preferably, at least one probe for identifying the kit is chosen from SEQ ID NO: 1 to SEQ ID NO: 4,
• microorganisms in the microbiological determination kit are chosen from the following bacteria:
• At least one probe for identifying the determination kit is chosen from SEQ ID NO: 14, SEQ ID NO 62, SEQ ID NO 66 to SEQ ID NO 69, SEQ ID NO 15, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 30, SEQ ID NO 9 to SEQ ID NO 11, SEQ ID 23, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70 % identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from the following viruses: Hepatitis A virus, Enterovirus, and at least one virus chosen from Caliciviruses and Rotaviruses.
• at least one probe of identification of the determination kit is chosen from SEQ ID NO 59, SEQ ID NO: 60 SEQ ID NO 97, SEQ ID NO 70 to SEQ ID NO 96, SEQ ID NO: 98 to SEQ ID NO: 104, and all fragments of these comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• Said microorganisms in the microbiological determination kit are chosen from the following viruses:
• at least one probe for identifying the determination kit is chosen from SEQ ID NO 98 to 104, SEQ ID NO 59, SEQ ID NO 56 to SEQ ID NO 58, SEQ ID NO: 60, SEQID NO: 97, SEQ ID NO: 70 to SEQ ID NO: 75, SEQ ID NO 76 to SEQ ID NO 96, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70 % identity with said any sequence.
• Said microorganisms in the microbiological determination kit are chosen from the following parasites: genus Cryptosporidium, Cryptosporidium parvum, genus Giardia, Giardia Lamblia.
• at least one probe for identifying the determination kit is chosen from SEQ ID NO: 40 to SEQ ID NO 45, SEQ ID NO 65, and all fragments thereof comprising at least 5 contiguous monomers included in one any of said sequences and the sequence of which has at least 70% identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from:
• SEQ ID NO 75 SEQ ID NO 76 to SEQ ID NO 96, SEQ ID NO: 40 to SEQ ID NO 45, SEQ ID NO 65, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• Said microorganisms of the microbiological determination kit are chosen from Norwalk virus, Hepatitis A virus, Enterovirus.
• at least one identification probe chosen from among SEQ ID NO 59, SEQ ID NO: 60, SEQ ID NO 97, SEQ ID NO 70 to SEQ ID NO 75.
• the capture probes advantageously comprise at least 10, preferably at least 13, or even at least 15, even at least 17 bases and / or at most 35, preferably at 25, or even at most 20.
• a capture probe has between 10 and 35 bases, advantageously between 17 and 20 bases, with at least one interrogation position located in the central region of the known sequence, in 12 th position relative to the 3 ′ end of the sequence.
• These capture probes have, depending on the case, lengths between 10 and 25 nucleotides. The interrogation positions then vary according to the length of the capture probe.
• identification sequences have been selected by computer selection techniques and are each sufficiently specific for a species and / or for a member of a species that they make it possible to discriminate between taxonomically close genera and / or species of the same genus and to avoid cross-hybridization phenomena.
• the microbiological determination kit comprises a mixture of bacteria and / or virus and / or parasite identification probes comprising at least four identification probes specific to at least four different bacteria. In another embodiment of the invention, the microbiological determination kit comprises a mixture of bacteria and / or virus and / or parasite identification probes comprising at least five identification probes specific to at least five different viruses .
• the microbiological determination kit comprises a mixture of bacteria and / or virus and / or parasite identification probes comprising at least two identification probes specific to a parasite.
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites comprising at least one probe specific for a bacterium and at least one probe specific identification of a parasite.
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites whose probes specific for bacteria are chosen from the probes specific for the following bacteria :
• Escherichia coli Genus Salmonella, Staphylococcus aureus.
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites whose probes specific for bacteria are chosen from the probes specific for the following bacteria :
• Escherichia coli Escherichia coli, genus Salmonella, Staphylococcus aureus, Clostridium perfringens.
• the microbiological determination kit comprises a mixture of identification probes bacteria and / or viruses and / or parasites whose specific probes for bacteria are chosen from the specific probes for the following bacteria:
• Escherichia coli Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium perfringens.
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites whose probes specific for bacteria are chosen from the probes specific for the following bacteria : Escherichia coli, Escherichia coli SEROTYPE 0157: H7,
• Helicobacter pylori Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium perfringens, Staphylococcus epidermitis, Staphylococcus aureus, Campylobacter coli, Campylobacter Aeri, Campylobacter aerospace , Acinetobacter baumanii, Burkholderia gladioli, Burkholderia cepacia, Stenotrophomonas maltophilia, the genus Mycobacteria, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium simiae, Mycobacterium kansasii, Mycobacterium genenella Legion, Mycobacterium marinum
• the microbiological determination kit comprises a mixture of bacteria and / or virus and / or parasite identification probes chosen from the following microorganisms: Escherichia coli, Genus Salmonella, Staphylococcus aureus, Genus
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites chosen from the following microorganisms:
• Genus Salmonella Staphylococcus aureus, Gardia lamblia, Cryptosporidium parvum.
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites chosen from the following microorganisms: Escherichia coli, - Enterococcus faecalis, Enterococcus faecium,
• Enterococcus durans Enterococcus durans, Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium perfringens, Genus Salmonella, Staphylococcus aureus,
• Enterovirus polio virus, coxsackie virus A and B, Echovirus, Genus Cryptosporidium, Cryptosporidium parvum, Giardia lamblia,
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites chosen from the following microorganisms: Escherichia coli, Enterococcus faecalis, Enterococcus faecium,
• Enterococcus durans Enterococcus hirae, Streptococcus bovis, Streptococcus equinus, Clostridium perfringens, Genus Salmonella, Staphylococcus aureus, Enterovirus: polio virus, coxsackie A and B virus, Echovirus, Genus Cryptosporidium, Legptia Giella , Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Campylobacter coli, Campylobacter jejuni, Hepatitis A virus, Calicivirus: Norwalk and Sapporo Virus, Adenovirus, Rotavirus.
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites chosen from the following microorganisms:
• the microbiological determination kit comprises a mixture of probes for identification of bacteria and / or viruses and / or parasites chosen from the following microorganisms: Escherichia coli, Enterovirus, Genus Cryptosporidium,
• the microbiological determination kit comprises a mixture of probes for identifying bacteria and / or viruses and / or parasites whose probes for specific identification of viruses are specific for the following viruses: Adenoviruses, such as Adeno virus 40, Adeno virus 41 bis; Astroviruses, HastV-1-2; Enteroviruses, such as Polioviruses, Coxsackieviruses, Echoviruses, Rotaviruses, Caliciviruses, such as Norwalk virus, Sapporo virus and, Hepatitis viruses such as Hepatitis A virus.
• Adenoviruses such as Adeno virus 40, Adeno virus 41 bis
• Astroviruses HastV-1-2
• Enteroviruses such as Polioviruses, Coxsackieviruses, Echoviruses, Rotaviruses, Caliciviruses, such as Norwalk virus, Sapporo virus and, Hepatitis viruses such as Hepatitis A virus.
• the microbiological determination kit comprises a mixture of bacteria and / or virus and / or parasite identification probes, the parasite specific probes of which are chosen from the following parasite specific probes : The genus Cryptosporidium, Cryptosporidium parvum, Giardia lamblia and Microsporidia.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 1 to SEQ ID NO: 39, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 66 to
• SEQ ID NO: 69 and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence; at least one identification probe chosen from SEQ ID NO: 40 to SEQ ID NO: 49, SEQ ID NO: 63 to SEQ ID NO: 65, and all fragments thereof comprising at least 5 contiguous monomers included in the any of said sequences and the sequence of which has at least 70% identity with said any sequence; and at least one sequence chosen from
• the kit for microbiological determination of a microorganism present in a sample comprises at least 4 identification probes chosen from SEQ ID NO: 1 to SEQ ID NO: 39, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 66 to SEQ ID NO: 69, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% d identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least 5 identification probes between SEQ ID NO: 50 to SEQ ID NO: 60, SEQ ID NO70 to SEQ ID NO: 104, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 40 to SEQ ID NO: 49, SEQ ID NO: 63 to SEQ ID NO: 65, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 1 to SEQ ID NO: 39, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 66 to SEQ ID NO: 69, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% d identity with said any sequence, and at least one identification probe chosen from SEQ ID NO: 40 to SEQ ID NO: 49, SEQ ID NO: 63 to SEQ ID NO: 65, and all fragments thereof comprising at least at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 15, SEQ ID NO: 23 , SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% d identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 66 , SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 28, SEQ ID NO: 29, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 66 , SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 40 to SEQ ID NO: 44, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 14, SEQ ID NO: 62, SEQ ID NO: 66 , SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 53 to SEQ ID NO: 55, SEQ ID NO70 to SEQ ID NO75, SEQ ID NO: 40 to SEQ ID NO: 44, and all fragments of those this comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one chosen identification probe. among SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 29, SEQ ID NO: 40 to SEQ ID NO: 49, SEQ ID NO: 53 to SEQ ID NO: 55, SEQ ID NO: 61 to SEQ ID NO75, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one identification probe chosen from SEQ ID NO: 1 to SEQ ID NO: 4, SEQ ID NO: 9 , SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14 to SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 29, SEQ ID NO: 40 to SEQ ID NO: 51, SEQ ID NO: 53 to SEQ ID NO: 55, SEQ ID NO: 56 to SEQ ID NO: 59, SEQ ID NO: 60 to SEQ ID NO: 65, and all fragments thereof including at at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one sequence between SEQ ID NO: 1 to SEQ ID NO: 6, SEQ ID NO: 9 to SEQ ID NO: 22, and SEQ ID NO: 23 to SEQ ID NO: 55, SEQ ID NO: 56 to SEQ ID NO: 104, and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one sequence chosen from SEQ ID NO: 14, SEQ ID NO 62, SEQ ID NO 66 to SEQ ID NO 69 , SEQ ID NO 15, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 30, SEQ ID NO 9 to SEQ ID NO 11, SEQ ID 23. and all fragments thereof comprising at least 5 contiguous monomers included in any of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one sequence chosen from SEQ ID NO 59, SEQ ID NO 97, SEQ ID NO 70 to SEQ ID NO 75 and all fragments of these comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one sequence chosen from SEQ ID NO 98 to 104, SEQ ID NO 59, SEQ ID NO 98, SEQ ID NO 56 to SEQ ID NO 58, SEQ ID NO 76 to SEQ ID NO 96 and all fragments thereof comprising at least 5 contiguous monomers included in any one of said sequences and the sequence of which has at least 70% identity with said any sequence.
• the kit for microbiological determination of a microorganism present in a sample comprises at least one sequence comprised between chosen from SEQ ID NO: 40 to SEQ ID NO 45, SEQ ID NO 65 and all fragments of these comprising at least 5 contiguous monomers included in any one of said sequences and whose sequence has at least 70% identity with said any sequence.
• identification sequences allow the selective assay of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• Adenoviruses such as Adeno virus 40, Adeno virus 41 bis; Astroviruses, HastV-1-2; Enteroviruses, such as Polioviruses, Coxsackieviruses, Echoviruses, Rotaviruses, Caliciviruses, such as Norwalk virus, Sapporo virus, and Hepatitis viruses, such as Hepatitis A, the genus Cryptosporidium, such as Cryptosporidium parvum , Giardia lamblia and Microsporidia.
• these identification sequences according to the invention allow the selective dosing of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• Escherichia coli Genus Salmonella, Staphylococcus aureus
• these identification sequences according to the invention allow the selective dosing of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• Escherichia coli Escherichia coli, genus Salmonella, Staphylococcus aureus, Clostridium perfringens.
• these identification sequences according to the invention allow the selective dosing of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• these identification sequences according to the invention allow the selective assay of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• these identification sequences according to the invention allow the selective dosing of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• these identification sequences according to the invention allow the selective dosing of a microorganism in the presence of at least 2 other microorganisms chosen from the following microorganisms:
• these identification sequences allow the selective dosing of a microorganism in the presence of at least 2 other microorgan
• microbiological determination kit comprising identification sequences to identify microorganisms at the level of serotypes, subtypes and in epidemiology.
• the method for analyzing a sample capable of containing at least one bacterium, parasite and / or virus, according to the invention uses a mixture of nucleotide sequences as identification probes specific for a serotype, a subtype , of a species or at least one kind of bacteria, virus and / or parasite likely to be present in the sample.
• This method of analyzing a sample according to the invention is characterized by a detection step comprising the implementation of a microbiological determination kit as defined above.
• At least one lysis step can be carried out prior to the detection step.
• an amplification step is carried out.
• the invention also relates to a method for controlling a liquid sample in which, prior to any detection step, a step of enriching said sample with microorganisms is carried out.
• This enrichment step can be carried out by filtration in particular using a filtration means comprising hollow fibers and used in frontal mode making it possible to obtain, in a limited time, from a starting liquid sample of large and predetermined volume, a sample to be analyzed of a sufficiently small volume, while guaranteeing the viability of the microorganisms, so that the analysis techniques, in particular multidetection according to the invention, can then be implemented.
• This filtration means is based on the technique of ultrafiltration on hollow fibers in frontal mode.
• frontal mode as opposed to tangential mode, is meant any passage in one pass of a starting liquid sample in the filtration means, without recycling at least a part of the same sample at the inlet of said filtration means.
• multi-recovery is meant the possibility of recovering from the final sample practically all the different genera or species of microorganisms present in the starting sample. These high yields are obtained due to the nonexistence of volumes, known as dead volumes, due for example on other devices to the presence of ancillary pipes, for example recycling, and by the reliability of the porosity over the entire length of hollow fiber.
• the control method according to the invention is thus applied to a sample possibly obtained by filtration, with a volume of between 1 ml and 100 liters.
• a step of lysis of the microorganisms is carried out, either by mechanical lysis or by chemical lysis, as described above.
• a purification step is optionally applied, optionally using capture techniques with oligonucleotides fixed on magnetic particles, or by using silica columns, silica particles (inert or magnetic), ion exchange columns, or any other method mentioned above.
• An enzymatic amplification step is also optionally applied, preferably using transcription techniques such as TMA, NASBA, but in particular using PCR and RT-PCR techniques.
• An amplicon labeling step is applied, by preferential use of a fluorescent label.
• a hybridization step is then applied, preferably using the specific identification probes or their fragments fixed on a solid support, and in particular using a biochip.
• control method and the microbiological determination kit according to the invention using these specific sequences makes it possible to simultaneously detect a bacterium and / or a virus and / or a parasite from a fixed panel in a single final multidetection step.
• Another subject of the invention is a process for the production and / or disinfection of a liquid, characterized in that it comprises an analysis step implementing a microbiological determination kit according to any one of Claims 35 to 48 and generating a data interpretation algorithm allowing the slaving of said production process and / or disinfection to said data generated by the microbiological determination kit.
• FIG. 1 represents the evolution of the base call on the specific probes of Escherichia coli and Acinetobacter baumanii as a function of the number of rRNA copies of each of the partners added before amplification.
• the area framed in the graph represents the E. coli / A proportions. baumanii from which E. coli targets can be interpreted by the chip.
• Figure 2 shows the evolution of base call on specific E probes. coli, S. typhimurium and A. baumanii according to the proportions of the number of copies of marked transcripts representing the 3 species.
• the strains used are:
• Example 1 Detection and identification of a single bacterial cell in culture: case of 1 Escherichia coli (gram -) and of Enterococcus faecalis (gram +)
• a lysis buffer containing 10mM Tris, 1mM EDTA (dilution of a TE100X solution sold by SIGMA, ref. T-9285) and lyzozyme (Sigma, ref. L-) are added 6876) whose concentration is different according to the Gram of the bacteria: 3 mg / ml for E. faecalis, 400 ⁇ g / l . for E. coli.
• the lysis of the bacteria is carried out by leaving the tube containing the bacterial suspension in contact with the lysis buffer for 5 to 10 minutes at room temperature.
• RT-PCR The two stages of RT and PCR will be carried out one after the other, in a single tube, using the ACCESS kit (ref A1250, Promega).
• the buffer is added to 25 ⁇ l of the total RNA suspension.
• the mixture is incubated for 45 minutes at 48 ° C., then 5 minutes at 94 ° C.
• 35 cycles are then carried out, each consisting of the following 3 steps: 94 ° C 1 min, 55 ° C 1 min, 68 ° C 1 min. A final extension of 7 minutes at 68 ° C is then carried out. d) Verification of amplification
• amplification product (amplicon) are deposited on a 1.5% agarose gel in EDTA-Tris Borate. After a migration of 20 minutes to
• the amplification band is visualized by staining with Ethidium Bromide and by UV illumination. It is shown that the amplification is positive by the presence of a band having the expected size (450 base pairs).
• e) Identification of the amplicon on a DNA chip (Affymetrix,
• a biochip is synthesized on a solid glass support according to the method described in US Pat. No. 5,744,305 (Affymetrix, Fodor et al) using the resequencing strategy described in application WO 95/11995 (Affymax,
• the oligonucleotides synthesized on the chip carry out the resequencing of the identification sequences. This process makes it possible to reduce the total number of oligonucleotides synthesized and therefore have a considerable advantage in terms of production cost and without compromising on the quality of the identification of the different microorganisms by the choice of these identification sequences.
• the oligonucleotides have 20 bases, with an interrogation position in 12th position relative to the 3 'end of the sequence. For the E.
• oligonucleotides of 17 bases with 2 interrogation positions: one in 10 and one in 8 th position.
• Other oligonucleotides have lengths between 10 and 25 nucleotides. The interrogation positions then vary as a function of the length of the oligonucleotide.
• the analysis is performed on the complete GeneChip® system (reference 900228, Affymetrix, Santa Clara, CA) which includes the GeneArray® reader, the GeneChip® hybridization oven, the GeneChip® fluid station and the GeneChip® analysis software. .
• the antisense primer S9T7 all the amplification products have a promoter for RNA polymerase T7. These amplicons will then serve as a matrix for a transcription reaction during which a fluorescent ribonucleotide will be incorporated.
• the labeled transcripts are cleaved into fragments of approximately 20 nuceotides.
• the 20 ⁇ l of labeled transcripts are subjected to the action of imidazole (SIGMA) 30 mM and manganese chloride (Merck) 30 mM for 30 min at 65 ° C.
• SIGMA imidazole
• Merck manganese chloride
• hybridization buffer the composition of which is SSPE 6X (Eurobio), DTAB 5 mM (Sigma), Triton 0.5% (Merk Eurolab).
• This mixture is hybridized on the chip under the following conditions: 40 min at 45 ° C. After washing, the chip is scanned, then the hybridization image obtained is analyzed by Genechip ⁇ software (Affymetrix, Santa Clara) The hybridization spots allow the amplicon sequence to be reconstructed, which is then compared to the sequences of chip references. The sequence (and therefore the species which corresponds to it) which has the best percentage of homology (base-call, in%) with the amplicon sequence is retained for identification.
• the interpretation threshold i.e. identification level, is set at at least 70% base-call on the identification sequence. Below this threshold, the target is not identified.
• RNA extracted from a single bacterial cell gives rise to an amplification product, then to a correct identification on the biochip.
• Example 2 Discrimination of mixtures of 2 different bacterial species.
• eubacterial RT-PCR was applied to synthetic targets. That is, these targets come from the amplification and then the transcription of the entire 16S ribosomal DNA. These targets are called in-vitro transcripts.
• the target is a mixture of in vitro transcripts representing the species Escherichia coli and Acinetobacter baumanii.
• we add the target to the RT-PCR tube we no longer resonate in terms of number of bacteria, but in number of copies of in vitro transcripts, then in number of bacteria equivalents, starting from the following assumption: 1 bacteria correspond to 10 4 copies of 16S ribosomal RNA.
• the transcripts were dosed at 10 11 copies / ⁇ l.
• the dilution 10 8 copies / ⁇ l is prepared.
• the dilutions 10 3 / ⁇ l, 10 4 / ⁇ l, 10 5 / ⁇ l and 10 6 / ⁇ l are prepared.
• the conditions of the reaction mixture for RT-PCR are identical to those described in Example 1, paragraph c), except that the target volume is no longer 25 ⁇ l of a suspension of total RNA, but 2 ⁇ l of a mixture consisting of 1 ⁇ l of each dilution of transcript representing each species in the proportions following:
• FIG. 1 shows that by reducing the number of copies of 16S rRNA to a number of bacteria, it is therefore possible to detect, by using the DNA chip, the equivalent of 1 E. coli in the presence of 10 4 In baumanii, a proportion of 0.01%.
• Example 3 Discrimination of a mixture of 3 different bacterial species
• the total number of copies in a hybridization mixture is fixed at 10 13 copies.
• transcripts corresponding to the E. coli and S. thyphimurium species are in the same number of copies. These transcripts have been added with respect to the transcripts of A. baumanii as follows:
• Figure 2 shows that the detection of E. coli is made at lower proportions (1%) than that of S. thyphimurium (10%). This result shows that it is possible to detect on the chip 3 different bacterial species.
• the strains are grown in Trypcase Soy broth at 37 ° C. When the culture reaches an optical density of 0.2-0.3 (10 8 bacteria / ml),
• the 3 bacterial species are mixed using the suspensions produced in part a), so as to have: 100 Escherichia
• the final volume will be 100 ⁇ l.
• RT-PCR is carried out using the ACCESS kit (Promega, ref.
• the base call on the identification sequence corresponding to each of the taxa must be greater than 90%. Below the target is not identified.
• Example 5 simultaneous detection of Escherichia coli, Staphylococcus aureus, Salmonella enteritidis and Pseudomonas aeruginosa
• Salmonella enteritidis ATCC 13076 Salmonella enteritidis ATCC 13076
• the bacterial suspensions are prepared according to the protocol indicated in Example 4, part a)
• the 4 bacterial species are mixed using the suspensions produced in part a), so as to have: 100 Escherichia coli, 100 Staphylococcus aureus, 100 Salmonella enteritidis and 100 Pseudomonas aeruginosa.
• RT-PCR is carried out using the ACCESS kit (Promega, ref. A1250) according to the protocol indicated in Example 1, part c).
• the base call on the identification sequence of each of the taxa must be greater than 90%. Below the target is not identified.
• Example 6 Simultaneous detection of Escherichia coli, Cryptosporidium parvum and Poliovirus Sabin 3.
• the RNeasy Mini Kit (Qiagen, ref. 74104) is used according to a modified protocol. To do this, 350 ⁇ l of RLT lysis buffer from the RNeasy kit is added to the 100 ⁇ l, and 25 ⁇ l of Proteinase K at 19 mg / ml (Roche, ref. 1964372) which brings back to 1 mg / ml. Leave to act for 30 minutes at 65 ° C. We then continue according to the RNeasy Mini Kit "for bacteria" protocol.
• Escherichia coli RT-PCR is carried out using the ACCESS kit (Promega, ref.
• RT-PCR is carried out using the ACCESS kit (Promega, ref. A1250). For this, we add to the 25 ⁇ l of total RNA obtained in step b) 2,
• reaction mixture so as to have, in the final 50 ⁇ l: the 1 ⁇ AMV / Tfl buffer, 2.5 mM MgSO4, the 200 ⁇ M dNTPs, 5 U of Tfl, 5 U of AMV, 5 U of RNAsin (Promega ref. N2111 ), and the primers XIA2F and XIA2R 200pM.
• Poliovirus Sabin 3 For the RT stage, the mixture is incubated for 45 min at 48 ° C. For the PCR step, incubate for 5 min at 94 ° C., then carry out 30 cycles each consisting of the following 3 steps: 94 ° C 45 sec, 55 ° C 45 sec, 68 ° C 1 min. A final extension of 7 min at 68 ° C is then carried out. 3. Poliovirus Sabin 3
• RT-PCR is carried out using the ACCESS kit (Promega, ref. A1250). For this, we add to the 25 ⁇ l of total RNA obtained in step b) 2,
• reaction mixture 25 ⁇ l of reaction mixture so as to have, in the final 50 ⁇ l: the AMV buffer Tfl 1X, the MgSO4 2 mM, the dNTP 300 ⁇ M, 5U of Tfl, 5U of AMV, 5U of RNAsin (Promega ref. N2111), and 200pM specific primers.
• the mixture is incubated for 45 min at 48 ° C.
• PCR step incubate for 2 min at 94 ° C, then 40 cycles are carried out, each of the following 3 steps: 94 ° C 15 sec, 55 ° C 30 sec, 68 ° C 1 min. A final extension of 7 min at 68 ° C is then carried out.
• the 3 tubes containing the 20 ⁇ l of transcription are combined the purification is done using the RNeasy Mini Kit (Qiagen ref. 74104), protocol for the purification of in vitro transcripts. 20 ⁇ l of transcript are obtained
• the base call on the signature sequence of E. coli and C. parvum must be greater than 90%.
• the detection threshold is above 85%
• Vaccine strain of the Hepatitis A virus at 17.5 DICC 50 / ⁇ L extraction of nucleic acids using the Qiamp Viral RNA kit from Qiagen -ref 52904- according to the supplier's instructions) 2- RT-multiplex PCR
• RT-PCR is carried out using the ACCESS kit (Promega, ref
• the mixture is incubated for 45 minutes at 48 ° C. After a denaturation stage of 2 minutes at 94 ° C, the
• Complementary DNA obtained are amplified in the following ways: 45 cycles of [15 seconds at 94 ° C, 30 seconds at 55 ° C, 45 seconds at 68 ° C] with an elongation step of 7 minutes.
• the base call on the sequence corresponding to each virus must be greater than 95%. Below this threshold, the target is not identified.

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