FR2995614A1 - Biochip used to detect Legionella bacteria in e.g. water sample, includes specific RNA polymerase beta subunit nucleic acid molecule that hybridizes with portion of sequence of subunit gene of bacteria and is immobilized on support - Google Patents

Abstract

Biochip comprises a specific RNA polymerase beta subunit (rpoB) nucleic acid molecule capable of specifically hybridizing with a portion of sequence of the rpoB gene of bacteria of a first species of Legionella or with its complementary sequence. The molecule is immobilized on a support. Independent claims are included for: (1) a kit for the detection and/or identification of bacteria of species of Legionella contained in a sample, comprising the biochip, a first pair of optionally labeled nucleic acid primers capable of amplifying the sequence of a region of rpoB gene of the bacteria comprising the portion of sequence with which the rpoB nucleic acid molecule specifically hybridizes; (2) detecting and/or identifying in vitro of bacteria of species of Legionella contained in a sample using the biochip, comprising amplifying the sequence of region of rpoB gene of the bacteria comprising the portion of sequence with which the rpoB nucleic acid molecule specifically hybridizes, contacting the amplified DNA with the biochip, and detecting possible hybridization complex; and (3) a set of nucleic acid primers.

Description

The present invention is in the field of microbiological diagnosis, specifically the detection and / or identification of Legionella bacteria in a sample likely to be contaminated by such bacteria. More particularly, the invention relates to a biochip adapted to such a detection, a diagnostic kit comprising such a biochip, and a method implementing it. Legionella bacteria (Legionella spp., Or Legionellae) are bacteria in the water environment that can cause a respiratory illness in humans, which can sometimes be fatal: Legionella, known as legionnaire's disease, characterized by severe acute pneumonitis. Legionnaire's disease is contracted by inhalation of water contaminated with legionella. These are often detected in water distribution systems and in devices that use water, such as air-cooling towers, air conditioning systems, nebulizers, humidifiers, etc. Twenty-one species of Legionella bacteria have so far been identified as pathogenic in humans, the most important being Legionella pneumophila (L. pneumophila) (O'Connell et al., 1996). Legionellosis is associated with a high mortality rate of about 20% (Meenhorst et al., 1985). It can especially be contracted in hospitals, most often through shower aerosols, respirators, or water used to humidify oxygen. Identifying cases of nosocomial infection is difficult, as the clinical spectrum of the disease ranges from mild influenza to severe pneumonia. In particular, no clinical or radiological argument makes it possible to differentiate with certainty legionellosis from other etiologies of pneumonia. However, an early diagnosis of this disease is essential to offer the patient a suitable antibiotic treatment. There are currently several methods for diagnosing infections caused by Legionella bacteria.

The cell culture technique is generally used to detect and quantify legionella in water samples. However, the time to obtain the results is long, and can reach ten days. In addition, this technique remains insensitive (10 to 30%) (Ballard et al., 2000, Boulanger and Edelstein, 1995), especially in the presence of interfering flora, inhibiting the growth of Legionella. Finally, legionella cells can exist in viable but non-culturable form, undetected by conventional culture techniques but potentially pathogenic (Steinert et al., 1997).

The diagnosis can also be made by direct immunofluorescence. Again, the technique lacks sensitivity and may cross-react with non-legionella isolates, which may make it difficult to identify other species (Bartie et al., 2003). The search for urinary antigens, specifically by an agglutination test of latex particles using antibodies specific for the outer cell membrane, is commonly used by hospitals, but is limited to the diagnosis of L. pneumophila ( von Baum et al., 2008). A test for detection and quantification of Legionella by Chain Polymerization Reaction (PCR) is also described in standard NF T 90-471. This test is currently commonly used to quantify L. pneumophila bacteria in air-cooling towers, as an alternative to culture techniques. The detection rate of Legionella DNA by PCR is generally high (more than 90% of positive samples) (Buchbinder et al., 2002, Ng et al., 1997), but the PCR positivity offers too little information on the risk of Legionnaires' disease. Thus, none of the techniques proposed by the prior art is entirely satisfactory, in particular because they are slow to implement, insensitive and / or limited to a given species. There remains therefore a need for a test for detecting, and preferably discriminating, Legionella bacteria in a sample that is quick to perform, and has high specificity and sensitivity.

The present invention thus aims to overcome the drawbacks of the detection methods of Legionella bacteria proposed by the prior art, especially those described above, by proposing a tool for the detection of at least one species of such bacteria in a sample, whether it is a biological sample from a patient suspected of having legionellosis, or a sample of water or air likely to be contaminated by such bacteria, particular of a species identified as pathogenic in humans, and a detection method using such a tool. The present invention more specifically aims at that such a tool and such a method of detection are based on the genotype of the bacteria, rather than on their phenotype, and that they have high levels of sensitivity and specificity. A further object of the invention is to allow the discrimination of different species of Legionella bacteria, and the identification, among a population of Legionella spp, of different species present, in particular of at least one species identified as pathogenic in the man. According to a first aspect, the present invention thus relates to a biochip adapted to the detection and / or identification of bacteria of at least one species of the genus Legionella given. This biochip comprises a nucleic acid molecule (also referred to by the term "probe" in the present description) called rpoB-specific, capable of hybridizing specifically with the sequence of a portion of the rpoB gene of bacteria of a first species of the genus Legionella, or with its complementary sequence, this molecule being immobilized on a support. Said species of the genus Legionella is preferably one of the species identified as pathogenic for humans, that is to say one of the following species: L. pneumophila, L. anisa, L. birminghamensis, L. bozemanii, L. cherrii , L. cincinnatiensis, L. dumoffii, L. erythra, L. feeleii, L. gormanii, L. hackeliae, L. jordanis, L. lansingensis, L. longbeachae, L. maceachemii, L. micadei, L. oakridgensis, L. Parisiensis, L. sainthelensi, L. tucsonensis, L. wadsworthii. Throughout the present description, the term "biochip" any system, preferably miniaturized, comprising a support on which are deposited, in particular covalently attached, nucleic acid molecules, called probes, each at a specific location of the support. The general principle of a biochip is to bring it into contact with a population of nucleic acids, called targets, and to detect any specific hybridization complexes of target nucleic acids with the probes immobilized on the support.

The support of the biochip according to the invention is conventional in itself, especially in the field of DNA chips. It can be solid or semisolid. It may for example be chosen from supports comprising at least one siliceous surface such as glass slides, beads and capillaries, and supports made of silicon, plastic or metal.

The rpoB-specific nucleic probe may be common to several species of Legionella, or to all species, exclusive of a given species. Such a biochip advantageously makes it possible to reliably and sensitively detect the presence, in a sample, of bacteria of at least one species of the genus Legionella, and, where appropriate, to identify the particular species to which these bacteria belong in the particular embodiments in which the rpoB-specific nucleic probe is exclusive of a given species. The invention thus advantageously takes advantage of the characteristics of the rpoB gene, which codes for the subunit 13 of the RNA polymerase, and which comprises a region of about 360 base pairs, called variable region, which, although strongly conserved between the different species of Legionella bacteria, allows a differentiation between many of these species. It is within the competence of those skilled in the art to identify, in the genomic databases, in particular in the Genbank database, the sequence of the rpoB gene belonging to each species of the genus Legionella given. Such sequences are described in particular for all 21 species identified as human pathogens, the Genbank accession number being, for each, indicated in parentheses after the name of the species: L. pneumophila (AF367748 strain ATCC 33152), L. anisa (AF367722, strain ATCC 35292), L. birminghamensis (AF367723, strain CIP 103871), L. bozemanii (AF367724, strain ATCC 33217), L. cherrii (AF367726, strain CIP 103842), L. cincinnatiensis (AF367727, strain CIP 103875), L. dumoffii (AF367728, strain ATCC 33279), L. erythra (AF367729, strain CIP 103843), L. feeleii (AF367731, strain CIP 103877), L. gormanii (AF367733, strain ATCC 33297), L. hackeliae (AF367735, strain CIP 103844), L. jordanis (AF367738, strain CIP 105268), L. lansingensis (AF367739, strain CIP 103542), L. longbeachae (AF367741, strain ATCC 33462), L maceachernii (AF367742, ATCC strain 35300), L. micadei (AF367743, ATCC strain 33218), L. oakridgensis (AF367746, strain CIP 103884 ), L. parisiensis (AF367747, strain CIP 103847), L. sainthelensi (AF367751, strain CIP 103885), L. tucsonensis (AF367756, strain CIP 105113), L. wadsworthii (AF367757, strain CIP 103886).

In particular embodiments of the invention, the biochip further comprises a molecule of nucleic acids (referred to by the term "probe" in the present description), said mip-specific, capable of hybridizing specifically with the sequence of a portion of the mip gene of bacteria of a species of the genus Legionella, which may be identical or preferably different from the first species, or with its complementary sequence, this molecule being immobilized on the support of the biochip. This mip-specific probe can also be as exclusive of a given species as it is common to several species of the genus Legionella, or even all.

The mip gene, which codes for the Mip protein, has already been described in the literature as permitting, by the presence of a variable region depending on the species, the discrimination between different species of Legionella bacteria (Ratcliff et al., 1998). ). Here again, those skilled in the art will be able to identify, in the genomic databases, in particular in the Genbank database, the sequence of the mip gene belonging to each species of the genus Legionella given. Such sequences are described in particular for all 21 species identified as human pathogens, the Genbank accession number being, for each, indicated in parentheses after the name of the species: L. pneumophila (S42595 strain ATCC 33152), L. anisa (U91607, strain ATCC 35292), L. birminghamensis (U91608, strain CIP 103871), L. bozemanii (U91609, strain ATCC 33217), L. cherrii (U91635, strain CIP 103842), L. cincinnatiensis (U91636, strain CIP 103875), L. dumoffii (U91637, strain ATCC 33279), L. erythra (U92203, strain CIP 103843), L. feeleii (U92205, strain CIP 103877), L. gormanii (U91638, strain ATCC 33297), L. hackeliae (U92207, strain CIP 103844), L. jordanis (U92209, strain CIP 105268), L. lansingensis (U92210, strain CIP 103542), L. longbeachae (X83036, strain ATCC 33462), L maceachemii (U92211, ATCC strain 35300), L. micadei (AF023175, ATCC strain 33218), L. oakridgensis (U92214, strain CIP 103884), L. parisiensis (U92215, strain CIP 103847), L. sainthelensi (U92219, strain CIP 103885), L. tucsonensis (U92224, strain CIP105113), L. wadsworthii (U92225, strain CIP 103886). In particularly preferred embodiments of the invention, the biochip comprises, immobilized on the support: a plurality of rpoB-specific nucleic acid molecules, capable of hybridizing specifically with different sequences respectively, by a variation of at least one nucleotide, portions, which may be located at the same or different loci, of the rpoB gene of bacteria belonging to the same species or to at least two different species of the genus Legionella, or with their complementary sequences, and a plurality of mip-specific nucleic acid molecules, capable of specifically hybridizing respectively with different sequences, by a variation of at least one nucleotide, of portions, which may be located at the same locus or at different loci, of mip gene of bacteria belonging to the same species or to at least two different species of the genus Legionella, which may be same or different from the one or more of the preceding ones, or with their complementary sequences, all of these nucleic acid molecules, that is to say rpoB-specific and mip-specific molecules, being chosen so that the The respective hybridization profiles of each species of the genus Legionella targeted with these molecules are different from each other.

Such an embodiment advantageously makes it possible to increase the sensitivity and the specificity of a test of detection, and if necessary of identification, of bacteria belonging to the same species or even to different species of the genus Legionella. For example, and not limitation, a biochip according to the invention may comprise: a first probe specific for a first sequence of a first portion of the rpoB gene of a first species; a second probe specific for a second species; sequence of a second portion of the rpoB gene of the first species, located at a different locus of the first portion; - a third probe specific for a sequence of a portion of the rpoB gene of a second species, this portion being able to or not be located substantially at the same locus as one of the preceding ones, - a fourth probe specific for a first sequence of a first portion of the mip gene of the first species, - a fifth probe specific for a sequence of a portion of the mip gene of a third species, this portion may or may not be located substantially at the same locus as the previous one, etc., each of these probes may or may not be exclusive of the associated species. In particular embodiments of the invention, the biochip comprises, immobilized on the support: at least one rpoB-specific nucleic acid molecule chosen from the nucleic acid molecules of respective sequences SEQ ID No. 1 to SEQ ID No. 7 or, for each of said sequences, any sequence having at least 95% identity with said sequence or with its complementary sequence, a sequence comprising it having not more than 25 more nucleotides or a fragment of at least 22 consecutive nucleotides included in said sequence, preferably a plurality, preferably all, of said molecules - preferably, at least one mip-specific nucleic acid molecule chosen from the nucleic acid molecules of respective sequences SEQ ID No. 8 at SEQ ID No. 17 or, for each of said sequences, any sequence having at least 95% identity with said sequence or with its sequence e complementary, a sequence comprising it comprising not more than 25 more nucleotides or a fragment of at least 22 consecutive nucleotides included in said sequence, preferably a plurality, preferably all, of said molecules. In the particular embodiment in which the biochip comprises all the nucleic acid molecules of respective sequences SED ID No. 1 to SEQ ID No. 17, or, for each of said sequences, any sequence having at least 95% d identity with said sequence or with its complementary sequence, a sequence comprising it comprising not more than 25 more nucleotides or a fragment of at least 22 consecutive nucleotides included in said sequence, this biochip quite advantageously makes it possible not only to detect the presence of bacteria belonging to each of the 21 species of Legionella genus identified as pathogens for humans, mentioned above, but also to discriminate these different species, and thereby to identify exactly which ones are present in a given sample this by implementing only a relatively limited number of different probes. The biochip according to such an embodiment of the invention makes it possible to perform a diagnostic test for the presence of legionella in a sample with particularly high levels of sensitivity and specificity. The nucleic acid molecule (s) immobilized on the biochip support may be of any nature or origin. It may especially be single-stranded or double-stranded nucleic acid molecules of natural or synthetic origin. In particular embodiments of the invention, the nucleic acid molecule, where appropriate each of the nucleic acid molecules, is an oligonucleotide obtained by chemical synthesis, by techniques known to those skilled in the art. These oligonucleotides preferably each have a length of about 25 to 30 nucleotides. Preferably, the respective melting temperatures of all the oligonucleotides of the biochip are very close, that is to say that the difference between the lowest and the highest melting point does not exceed 8.2 ° C. . In particular embodiments of the invention, each molecule of nucleic acids is immobilized on the support of the biochip by means of phosphorus dendrimers having a central core containing at least two functional groups and having at their periphery a first function for their covalent attachment to the support and a second function for covalent attachment of the nucleic acid molecule. The biochips thus formed advantageously have excellent stability and a particularly important detection sensitivity. They are also easy to manufacture, in a few steps, in a controlled and reproducible manner, at a low cost.

These dendrimers preferably have a size of between 1 and 20 nm. In general, dendrimers are isomolecular hyperbranched polymers whose size, topology and molecular weight can be rigorously controlled during their formation. The dendrimer molecule, globally spherical from a certain size, results from the repetitive radial branching of monomers from a central core. The biochips whose spacer arms are dendrimers advantageously have a high sensitivity and an improved signal-to-noise ratio compared to other spacer arms, in particular because the dendrimers make it possible to obtain a high density of probes per unit area of the support, and better accessibility of the probes, which results in hybridization with the target DNA, which is not two-dimensional, but three-dimensional.

Preferably, the dendrimers are chosen from those consisting of: a central layer in the form of a central core Po, optionally phosphorus, comprising from 2 to 12 functionalized groups, of n intermediate layers, identical or different, each of said layers intermediates consisting of units P1 corresponding to the following formula (I): in which: L is an oxygen, phosphorus, sulfur or nitrogen atom, M represents one of the following groups: an aromatic group di-, tri- or tetrasubstituted with alkyl groups, alkoxy groups, unsaturated groups of the olefinic group C1-C12, azo, acetylenic, all of which groups may or may not incorporate phosphorus, oxygen, nitrogen atoms , sulfur or halogen, or - an alkyl or alkoxy group having more than one substituent as defined when M is an aromatic group, R1 and R2, which may be the same or different, represent a hydrogen atom or one of the following groups: alkyl, alkoxy, aryl, with or without phosphorus, oxygen, sulfur, nitrogen or halogen atoms with R2 being most often different from R1, n is an integer from 1 to 11, E is an oxygen, sulfur or nitrogen atom, said nitrogen atom being bondable to an alkyl, alkoxy or aryl group, all of which may be whether or not to incorporate phosphorus, oxygen, nitrogen, sulfur or halogen atoms, of an external layer consisting of identical or different P2 units, and corresponding to the following formula (II): which: W represents one of the following groups: alkyl, alkoxy, aryl, all these groups optionally comprising atoms of phosphorus, oxygen, nitrogen, sulfur or halogens, X represents an aldehyde, thiol group, amine, epoxide, carboxylic acid, alcohol or phenol.

In preferred embodiments of the invention, X represents an aldehyde group. Microarray supports functionalized with such dendrimers are described in particular in patent document WO 03/091304.

Biochips in accordance with the invention may be prepared by carrying out the following schematic steps, by conventional chemical synthesis techniques in themselves: - if appropriate, silanization of the support, in a prior stage, - fixation on the support, preferably by covalent bonding, of a spacer arm, in particular of a phosphoric dendrimer as defined above, and attachment to the spacer arm, preferably by covalent bonding, probes modified to present a functional group at one end , for example the 5 'end.

According to a second aspect, the present invention relates to a kit for the detection and / or identification of bacteria of at least one species of the genus Legionella contained in a sample, comprising: a biochip responding to one or more of the characteristics above, and a first couple of nucleic primers, optionally labeled, capable of amplifying the sequence of a region of the rpoB gene of said bacteria comprising the portion (s) with the sequence (s) of which of which the rpoBspecific nucleic acid molecule (s) is / are capable of hybridizing specifically.

These nucleic primers are preferably oligonucleotides obtained by chemical synthesis. In particular embodiments of the invention, these primers are chosen to be able to hybridize with rpoB gene sequences, or flanking sequences, which are conserved for all species of the genus Legionella, or at least for 21 pathogenic species for humans. In preferred embodiments of the invention, this first pair of nucleic primers consists of primers A1 and A2, sequences SEQ ID No. 18 and SEQ ID No. 19 respectively, or any pair of primers whose The respective sequences have at least 90%, preferably at least 95%, identity with these sequences SEQ ID No. 18 and SEQ ID No. 19 or with their complementary reverse sequences. Such pairs of primers, corresponding to conserved sequences for all Legionella species, quite advantageously make it possible to amplify the sequence of a region of the rpoB gene of the bacteria which comprises the variable region making it possible to discriminate between the various species of the species. genus Legionella. This region notably comprises the sequences SEQ ID No. 1 to SEQ ID No. 7 of the rpoB-specific nucleic probes described above. These primers make it possible to amplify an approximately 360 base pair DNA fragment of the rpoB gene, regardless of the species of bacteria of the genus Legionella, and in particular the pathogenic species for humans. Preferably, the kit further comprises a second pair of optionally labeled nucleic primers capable of amplifying the sequence of a region of the mip gene of said bacteria comprising the portion (s) with the sequence (s) of which / or the mip-specific nucleic acid molecule (s) is / are capable of hybridizing specifically. The nucleic primers of this second pair are also preferably oligonucleotides obtained by chemical synthesis. Preferably, these primers are chosen to be able to hybridize with mip gene sequences, or flanking sequences, which are conserved for all species of the genus Legionella, or at least for the 21 pathogenic species for humans.

In particular embodiments of the invention, this second pair of nucleic primers consists of primers A3 and A4, sequences SEQ ID No. 20 and SEQ ID No. 21 respectively, or any pair of primers whose The respective sequences have at least 90%, preferably at least 95% identity with these sequences SEQ ID NO: 20 and SEQ ID NO: 21 or with their complementary reverse sequences. In the same way as described above for the rpoB gene, such pairs of primers, corresponding to conserved sequences for all Legionella species, quite advantageously make it possible to amplify the sequence of a region of the mip gene. bacteria that includes the variable region to discriminate different species of the genus Legionella. This region notably comprises the sequences SEQ ID No. 8 to SEQ ID No. 17 of the mip-specific nucleic probes described above. These primers make it possible to amplify a DNA fragment of about 600 base pairs of the mip gene, irrespective of the species of bacteria, and in particular the pathogenic species for humans. Preferably, at least one nucleic primer of each of the above pairs is labeled with a conventional detectable label in itself, in particular with a fluorescent label, such as a fluorophore, for example at its 5 'end, so as to generate a detectable and, where appropriate, quantifiable signal. The kit may further comprise all or part of the reagents necessary for the implementation of the DNA amplification of the sample, in particular by PCR, and / or the hybridization of the amplified DNA with the probes of the sample. biochip. It also preferably includes instructions for use, including in particular the hybridization temperatures recommended for the primers and / or for the probes. A third aspect of the invention is a method of detecting and / or identifying in vitro bacteria of at least one species of the genus Legionella contained in a sample, by means of a biochip responding to one or more characteristics above. This method comprises: amplifying a sequence of a region of the rpoB gene of the bacteria contained in the sample, this region comprising the portion (s) with the sequence (s) from which the or the rpoB-specific nucleic acid molecule (s) is / are capable of hybridizing specifically, by contacting with a first pair of specific, possibly labeled nucleic primers, preferably by polymerase chain reaction (PCR), - bringing the amplified DNA into contact with the biochip, and - detecting a possible hybridization complex between a DNA molecule thus amplified and a nucleotide probe rpoB- specific biochip.

The sample may be of any type, in particular consisting of a sample of water or air likely to be contaminated by bacteria of the genus Legionella, or a biological sample from a patient suspected of having legionellosis, for example from a usual collection for this type of infections, such as a nasopharyngeal swab, sputum, etc.

This method is preferably carried out on a population of nucleic acids previously extracted from the bacteria contained in the sample, by techniques known to those skilled in the art, in particular by lysis of the cells followed by purification of the DNA contained therein. Preferably, the amplification of a sequence of a region of the rpoB gene of the bacteria is carried out by means of a pair of nucleic primers consisting of primers A1 and A2, with sequences SEQ ID No. 18 and SEQ ID No. 19, or any pair of primers whose respective sequences have at least 90%, preferably at least 95%, identity with said sequences SEQ ID No. 18 and SEQ ID No. 19 or with their complementary reverse sequences. In particular embodiments of the invention, the method further comprises: amplifying a sequence of a region of the mip gene of the bacteria of the sample, this region comprising the portion (s) ) with the sequence (s) of which the mip-specific nucleic acid molecule (s) is / are capable of hybridizing specifically, by contacting with a second couple of specific nucleic primers, possibly labeled, preferably by PCR, - bringing the thus amplified DNA into contact with the biochip, and - detecting a possible hybridization complex between a DNA molecule thus amplified and a mip-specific nucleic probe of the biochip. Preferably, the amplification of a sequence of a region of the mip gene of the bacteria is carried out by means of a pair of nucleic primers consisting of primers A3 and A4, sequences of SEQ ID No. 20 and SEQ ID No. 21, or any pair of primers whose respective sequences have at least 90%, preferably at least 95%, identity with said sequences SEQ ID No. 20 and SEQ ID No. 21 or with their complementary reverse sequences. In preferred embodiments of the invention, the amplification of the sequence of a region of the rpoB gene and the amplification of the sequence of a region of the mip gene of the bacteria are carried out by a reaction method of multiplexed chain polymerization, wherein the first pair of primers and the second pair of primers are implemented simultaneously.

The method may further comprise a step of identifying species of the Legionella genus present in the sample, by comparing the hybridization profile obtained on the biochip with a pre-established matrix mapping each immobilization site of a nucleic probe. on the biochip, with the species or species of which said probe is specific. The various stages of comparison and deduction of the species present in the sample are preferably carried out by a programmed computer type computer, comprising at least one microprocessor, and storage means (magnetic hard disk, flash memory, optical disk, etc. .) in which is stored a computer program product, in the form of a set of program code instructions to be executed to implement, from information transmitted by a detector, signals generated at each site of the biochip subsequent to the formation of a hybridization complex, and from said predetermined matrix, the various steps of comparison and deduction of the method according to the invention. The method according to the invention advantageously makes it possible to carry out an in vitro diagnosis of the presence of Legionella bacteria in a sample which is reliable and accurate. This method is also simple, fast and inexpensive to implement, and highly reproducible. In particular, it makes it possible, in preferred embodiments, to detect and discriminate the 21 species of Legionella bacteria identified as pathogens for humans. According to a fourth aspect, the present invention relates to the use of a biochip responding to one or more of the above characteristics, for the detection and / or identification of bacteria of at least one species of the genus Legionella, preferably a plurality of different species of the genus Legionella, and in particular of the 21 pathogenic species for humans. The present invention also relates to a set of nucleic primers comprising the following pairs of primers: primers A1 and A2, with sequences of SEQ ID No. 18 and SEQ ID No. 19, respectively, or any pair of primers whose respective sequences have at least 90%, preferably at least 95%, identity with said sequences SEQ ID No. 18 and SEQ ID No. 19 or with their complementary reverse sequences, these primers being able to together amplify a sequence of a region about 360 base pairs of the rpoB gene of the bacteria, whatever the species to which they belong, this region being able to discriminate certain species from each other, - primers A3 and A4, of sequences respectively SEQ ID No. 20 and SEQ ID No. 21, or any pair of primers whose respective sequences have at least 90%, preferably at least 95%, identity with said SEQ ID NO: 20 sequences. and SEQ ID NO: 21 or with their complementary reverse sequences, these primers being able to together amplify a sequence of a region of about 600 base pairs in length of the mip gene of bacteria, whatever the species to which they belong, this region being able to discriminate certain species from each other, at least one primer of each pair being preferentially marked, in particular by a fluorescent marker. The features and advantages of the invention will appear more clearly in the light of the example below, provided for illustrative and not limiting purposes of the invention, with the support of FIG. 1, which shows the image , obtained at a wavelength of 635 nm by a laser fluorescence reader, of a biochip according to one embodiment of the invention after contacting with DNA obtained by PCR amplification, by means of pairs of d respectively rpoB gene and mip gene specific primers, from a sample containing bacterial strains of the species L. cincinnatiensis, L. longbeachae and L. birminghamensis. A / Biochip A biochip according to a particular embodiment of the invention is prepared as follows. The steps for preparing the phosphorus dendrimers and for preparing and functionalizing the support with such dendrimers are described in particular in patent document WO 03/091304 and the publications by Trévisiol et al., 2003 and Le Berre, 2003. 25 1 / Preparation Phosphorus dendrimers are prepared as follows DP4 phosphorus dendrimers, corresponding to the general formula (III): ## STR1 ## In a first step, the N-methyldichlorothiophosphorhydrazide (IV), a primordial synthon for obtaining the dendrimer, according to the reaction scheme: ## STR2 ## ## STR2 ## This is achieved by adding dropwise, under argon, a solution of methylhydrazine (1.9 equiv.) In chloroform CHCl3, on a solution of trichlorothiophosphine equiv.) In chloroform, keeping the temperature of the mixture at -60 ° C throughout the addition. The mixture is then slowly allowed to rise to room temperature overnight, while maintaining stirring. The following day, the reaction is monitored by 31 P {1 H} NMR and left stirring if necessary for one to two more days. The monomethylhydrazine hydrochloride obtained is then filtered under argon using a filtering cannula. The N-methyldichlorothiophosphorhydrazide is kept in solution in chloroform at low temperature (-20 ° C) and is used as such thereafter. In the next step, the free aldehyde end dendrimer (V), precursor of the phosphorus (III) dendrimers is prepared: (V) To this end, are mixed in a flask under argon, hexachlorocyclotriphosphazene ( 1 equiv), 4-hydroxybenzaldehyde (6.6 equiv) and distilled THF, taken under argon. This mixture is stirred until the solids have dissolved completely. Potassium carbonate (12 equiv) is then added spatula per spatula, and the mixture is left stirring overnight at room temperature. The following day, the reaction is monitored by 31 P {1 H} NMR. The potassium carbonate is filtered on filter paper and the filtrate is concentrated on a rotary evaporator to give a white solid. At room temperature, the solid is taken up in methanol, filtered on sintered and rinsed twice with methanol and twice with ether. The dendrimer is then obtained corresponding to the general formula (V) above, called generation 0, named DPO. The phosphoric dendrimer used to functionalize the so-called fourth generation glass slides, named DP4, corresponding to the general formula (III) above, is then obtained by repetition of the same sequence of two reactions, and this up to to obtain the 4th generation: 1 / Under argon, are mixed DPn (n representing the generation of dendrimer, and n = 0 to 3) (1 equiv), CHCI3 and the solution of N-methyldichlorothiophosphorhydrazide prepared as described above (7, 13, 27 and 53 equiv., respectively).

After 2 h stirring for small generations, and 3 h for large, at room temperature, the reaction is monitored by 31 P {1 H} NMR. The mixture is concentrated by half under reduced pressure, using a rotary evaporator, transferred into a dropping funnel and added dropwise to a large volume of pentane to precipitate the product. The precipitate is filtered using a cannula. The solid is taken up in a minimum of chloroform, precipitated again in a pentane / diethyl ether mixture: 4/1 and filtered using a cannula. Dendrimers with chlorine ends of 1st, 2nd, 3rd and 4th generation are thus obtained, named respectively DP'1, DP'2, DP'3, DP'4. 2 / Under an argon atmosphere and at ambient temperature, DP'n (n = 1 to 4) (1 equiv.) And 4-hydroxybenzaldehyde (13, 28, 55 and 110 equiv., Respectively) are introduced, then distilled THF taken under argon. Cesium carbonate (20, 40, 60 and 120 equiv., Respectively) is then added spatula per spatula. The mixture is stirred at room temperature for 16 h (overnight). The following day the reaction is monitored by 31 P {1 H} NMR. The salts are removed by filtration on filter paper for small generations and then using the centrifuge, and the filtrate is evaporated under reduced pressure to give a white solid. The solid is solubilized in a minimum of chloroform and added dropwise to a large volume of a pentane / ether mixture in order to precipitate the product. The precipitate is filtered on sintered. The solid is taken up in chloroform, precipitated again and filtered. The dendrimers with aldehyde ends of the 1st, 2nd, 3rd and 4th generation, named DP1, DP2, DP3 and DP4 are thus obtained. 2 / Preparation and functionalization of the support The support used consists of a glass slide modified by the following steps: - Silanization: the slide is first washed in a 2.5 M solution of alcoholic sodium hydroxide (NaOH in a H2O milliQ / EtOH 96%) for 2 hours. After returning to neutrality by 3 successive washings with milliQ water, the slide is immersed in 96% ethanol (for at least 5 min and at most 2 h), then it is immersed in the silanization bath comprising 3'-aminopropyltrimethoxysilane (APTES) in 96% ethanol EtOH. The slide is left in this bath for 1 night with stirring at room temperature. It is then left in the open air for 30 min, then rinsed several times with milliQ water, one time using an ultrasonic bath to remove the residues stuck on the blade, and dipped in an EtOH bath. 96%, then absolute EtOH, before being dried by centrifugation. The slide is finally held at 120 ° C for 3 hours to ensure crosslinking of the silane coating on the slide. Functionalization with dendrimers: the slide is immersed in a solution of DP4 dendrimers, prepared as described above, at 58 μM in tetrahydrofuran (THF), for 6 hours at room temperature. It is then rinsed once with THF, once with 96% EtOH and finally with absolute EtOH, before being dried by centrifugation. 3 / Immobilization of the nucleic probes on the support The nucleic probes used are indicated in Table 1 below, with in particular, for each, its sequence and the associated target gene. These probes are oligonucleotides obtained by chemical synthesis, by conventional techniques in themselves. Their sequences are specific to Legionella spp., More specifically, for each of them, at least one particular species of the genus Legionella. In general, for each probe, an alignment of sequences with the target gene sequence rpoB or mip of each species of interest available in the genomic databases, in particular the Genbank database, makes it possible to know which (s) Species (s) it is specific.

Designation Sequence (5 '.... 3') SEQ ID No. Target gene R1 AAAGAACGTTTAAGTTTGGTTGAGT 1 rpoB GGTTAAAGAACGTTTAAGTTTGGTT R2 2 R3 R4 GTTTAGAGTAAGTCTTGTTCGTGTTG ACAGAAAACCAATTTAGAGTTGGTCTTG 3 4 5 GAAAGAGCAGTTAAAGAACGATTAAG R5 R6 R7 GGATCAAGTAAACCCATTATCAGGG GTTAAAGAGCGATTGAGTTTAGTTG 6 7 8 mip GAAGAGAACAAGGCTAAAGGTGAAG M1 M2 M3 AAAAGCAGAAGAAAACAAAGCAAAA 9 AAAAGCTGAAGAAAACAAAGCTAAG 10 GAAGAAAATAAAGCGAAAGGTGAAG M4 M5 11 12 M6 GAAGAAAACAAGATTAAAGGCGAAG GAAGAGAATAAGTCGAAAGGAGAAG GAAGACAATAAGGCAAAAGGAGAAG M7 13 M8 14 M9 15 AAAATCTGAAGAAAACAAAGCAAAA GAAGAAAACAAAGCAAAAGGTGAAA CTGTCCCAGAACAAAACCAAAGAAG M10 16 17 Table 1 - Description of nucleic acid probes attached to the biochip These probes all have a melting temperature Tm of between 54.7 and 62.90 ° C. These probes are deposited on the support functionalized by the dendrimers in the form of modified C6-NH2 oligonucleotides, at a concentration of 20 μM, in a 0.1 M phosphate buffer at pH 9, using a needle-type robot. Q-Array Mini (Genetix), any other robot capable of performing such a deposit that could otherwise be implemented.

A reduction is then performed to consolidate the covalent bond between the free aldehyde of a dendrimer and the NH 2 end of each oligonucleotide. The slide is soaked for 3 h in a solution of NaBH4 3.5 mg / ml, then washed 3 times for 5 min in water milliQ, and finally dried by centrifugation Each probe is deposited in triplicate, c ' that is to say at 3 immobilization sites different from the blade, to ensure the reliability of the result of the hybridizations. A matrix is established recording the location of each probe on the blade.

B / Diagnostic kit A diagnostic kit for the identification of the species of bacteria of the genus Legionella present in a sample, according to a particular embodiment of the invention, comprises: the biochip described above, the first next pair of primers, to amplify a sequence of about 360 base pairs of the rpoB gene of the bacteria: Al (sense): 5'-GATGATATCGATCAYCTDGG-3 '(SEQ ID NO: 18) A2 (antisense): 5 VGGCGTTTCAATNGGAC-3 '(SEQ ID NO: 19) wherein Y is C or T, D is A or G or T, V is A or C or G, and N is A or T or G or C - the second next primer pair, for amplifying a sequence of about 600 base pairs of the bacteria mip gene: A3 (sense): 5'-TTTATGAAGATGARAYTGGTCRCTGC-3 '(SEQ ID NO: 20) A4 (antisense): 5 ## STR2 ## wherein Y is C or T, D is A or G or T, R is prepared according to conventional oligonucleotide synthesis techniques. More precisely, each corresponds to an equimolar mixture of several oligonucleotides of different sequences, each corresponding to one of the possible combinations of the nucleotides represented by the letters Y, D, V, N and R.

Each of the antisense primers A2 and A4 is further labeled with a fluorescent label, more particularly by the fluorophore Cy5, at its 5 'end. Here again, the marking is carried out by techniques known to those skilled in the art. These primers all have a melting point Tm of between 47.7 and 57.1 ° C. C / In Vitro Diagnostic Procedure 1 / Preparation of the Sample An aqueous sample comprising bacteria of the species L. cincinnatiensis, L. longbeachae and L. birminghamensis is prepared in the following manner. These species have the characteristics listed in Table 2 below. Species L. cincinnatiensis L. longbeachae L. birminghamensis CIP strain 103875 ATCC 33462 CIP 103871 accession number Genbank AF367727.1 AF367741.1 AF367723.1 of the rpoB gene (GI: 21702419) (GI: 21702447) (GI: 21702411) (SEQ ID No.) (SEQ ID NO: 22) (SEQ ID NO: 24) (SEQ ID NO: 26) GenBank U91636.1 accession number X83036.1 U91608.1 of the mip gene (GI: 2231689) (GI: 897774) (GI: 2231683) (SEQ ID NO:) (SEQ ID NO: 23) (SEQ ID NO: 25) (SEQ ID NO: 27) mip gene region 430-980 548-1098 38-585 amplified by primers A3 and A4 Species-specific biochip probes R3, R5, M2, M9 R3, R5, R6, M9 M8, M9 Table 2 - Characteristics of Species in the Specimen For each of the species above , primers A3 and A4 make it possible to amplify the sequence of the mip gene between the nucleotide positions of the respective sequences SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 27, indicated in Table 2. The primers A1 and A2 are able to hybridize on regions of the rpoB gene surrounding the sequences SEQ ID No. 22, SEQ ID No. 24 and SEQ ID No. 26 respectively. For each of the 3 species, for each of the rpoB and mip genes, the region amplified by the primers comprises the hybridization sites of the associated probes indicated in Table 2. Each strain is cultured for 48 to 72 hours at 37 ° C. 10 on a medium BCYE (for English Buffered Charcoal Yeast Extract). A colony of each strain is removed and seeded in 200 .mu.l of water. The three suspensions thus obtained are mixed at equal concentrations to form the sample on which a process according to a particular embodiment of the invention will be applied. 2 / Extraction of DNA from the sample For each strain, the DNA is extracted using a "High Pure PCR Template Preparation Kit" kit (Roche Applied Science). 3 / DNA Amplification The amplification and labeling of the extracted DNA is carried out by multiplex PCR in a total volume of 50 μL. The reagents used are as follows: 35 μl of Buffon OX (Sigma-Aldrich), 2 μM of MgCl 2 (Sigma-Aldrich), 200 μM of a dNTP mix (Sigma-Aldrich), 1,411 μl of each of the Al primers. and A2, 111M of each of A3 and A4 primers and 2.5 U of JumpStart® Taq DNA polymerase (Sigma-Aldrich). These reagents are mixed with 5 μl of DNA extracted from the strains in 0.2 ml PCR microtubes. The reaction is carried out in a GTQ-Cycler 96 thermal cycler (Hain LifeScience), according to the following program: an initial denaturation step and activation of the enzyme for 1 minute at 94 ° C., 40 amplification cycles [ denaturation for 10 seconds at 94 ° C., hybridization for 20 seconds at 54 ° C., elongation for 30 seconds at 72 ° C.), final elongation for 1 minute at 72 ° C. After amplification, the amplification products obtained are purified using the kit "MinElute® PCR Purification Kit" (Qiagen). The DNA thus amplified and labeled, and purified, is denatured for 2 minutes at 94 ° C and then immediately incubated in ice to avoid reformation of the double helix. 4 / Hybridization of DNA on the biochip All the purified PCR product is then mixed with 70 μl of hybridization buffer (5X Denhardt solution (Invitrogen), 5 × SSC (Sigma-Aldrich), 10 μg of salmon sperm DNA (Invitrogen, final concentration 100 μg / ml), H20 qsp 100 μl). The homogenized mixture is deposited on the biochip, which is then incubated for 30 minutes at 60 ° C., so as to allow the possible hybridization of DNA with the probes of the biochip. The non-specific hybridizations are then removed by the following three successive washings, carried out at room temperature and with stirring, by successive immersion of the biochip in 40 ml of: - washing buffer T1 [2X SSC (Sigma-Aldrich), 0, 2% SDS (Fluka)], for 3 minutes, - again washing buffer T1, for 3 minutes - washing buffer T2 [0.2 X SSC], for 3 minutes. The biochip is then dried by centrifugation. 5 / Detection of the hybridization complexes After possible hybridization of DNA of the sample with one or more probes of the chip, the possible hybridization complexes are revealed by reading the fluorescence at 635 nm (resolution 10 μm) with a laser type reader. The image shown in FIG. 1 is obtained, in which each point indicated in the clear represents a probe recognized by the DNA extracted from the sample. A fluorescence value is assigned to each probe of the biochip, establishing the associated signal-to-noise ratio. For each probe deposited on the triplicate plate, the average of the three values is calculated, to obtain the relative value Vh characterizing the hybridization of DNA on each probe. The results given in Table 3 below are obtained. Probe R1 R2 R3 R4 R5 R6 R7 M1 M2 Vh 0 0 1125 0 5 1254 0 0 12 Probe M3 M4 M5 M6 M7 M8 M9 M10 Vh 0 0 0 0 0 10 34 0 Table 3 - Relative value Vh characterizing the hybridization of each probe with the DNA extracted from the sample The hybridization profile of the sample thus obtained is compared to a matrix which associates the probes with the different species of bacteria of the genus Legionella. It is deduced that the sample contains the following species: L. cincinnatiensis, L. longbeachae and L. birminghamensis. These are the species initially present in the sample. Such a method according to a particular embodiment of the invention, simple and quick to implement, advantageously allows to detect and discriminate many species of Legionella, including the 21 species identified as pathogens for humans listed below. before, even if these species are mixed with each other or with other microorganisms. This process is highly reproducible, and has high levels of sensitivity and specificity. It can also be implemented, at least for the final stages of data processing and obtaining results, in an automated manner.

BIBLIOGRAPHIC REFERENCES Ballard et al., 2000. Detection of Legionella pneumophila using a real-time PCR hybridization assay. J. Clin. Microbiol. 38, 4215-4218 Bartie et al., 2003. Methods for Legionella from environmental 5 samples. Water Res. 37, 1362-1370 Baker and Edelstein, 1995. Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation. Apt. About. Microbiol. 61, 1805-1809 Buchbinder et al., 2002. Evaluation of detection of Legionella spp. in situ fluorescence in situ hybridization, PCR amplification and bacterial culture. Int. J. Med. Microbiol. 292, 241-245 Le Berre et al., 2003. Dendrimeric coating of glass slides for sensitive DNA microarrays analysis. Nucleic Acids Research 31, 88th-88 Meenhorst et al., 1985. Water-related nosocomial pneumonia caused by Legionella pneumophila serogroups 1 and 10. J. Infect. Dis. 152, 356-364 Ng et al., 1997. Comparison of polymerase chain reaction and the theory of detection in Singapore. Lett. Apt. Microbiol. 24, 214-216 O'Connell et al., 1996. Infection of Macrophage-like Cells by Legionella Species that have not been associated with disease. Infect. Immun. 64, 4381-4384 Ratcliff et al., 1998, Journal of Clinical Microbiology, Vol. 36, No. 6, 1560-1537 Steinert et al., 1997. Resuscitation of Viable Nonculturable Legionella Pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Apt. About. Microbiol. 63, 2047-2053 Trévisiol et al., 2003. Dendrislides, dendrichips: a simple chemical functionalization of glass slides with phosphorus dendrimers as an effective means for the preparation of biochips. New Journal of Chemistry 27, 1713 von Baum et al., 2008. Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin. Infect. Dis. 46.1356-1364

Claims (19)

REVENDICATIONS1. A biochip comprising a nucleic acid molecule called rpoBspecific, capable of hybridizing specifically with the sequence of a portion of the rpoB gene of bacteria of a first species of the genus Legionella, or with its complementary sequence, said molecule being immobilized on a support. 2. A biochip according to claim 1, comprising a mip-specific nucleic acid molecule capable of hybridizing specifically with the sequence of a portion of the mip gene of bacteria of a species of the genus Legionella, preferably different from said first species, or with its complementary sequence, said molecule being immobilized on said support. 3. A biochip according to any one of claims 1 to 2, comprising, immobilized on said support: a plurality of rpoB-specific nucleic acid molecules, capable of hybridizing specifically respectively with different sequences of portions of the rpoB gene bacteria belonging to the same species or to at least two different species of the Legionella genus, or with their complementary sequences, and a plurality of mip-specific nucleic acid molecules, capable of hybridizing specifically with different sequences of portions of the mip gene of bacteria belonging to the same species or to at least two different species of the genus Legionella, or with their complementary sequences, all of said nucleic acid molecules being chosen such that the hybridization profiles of each said species of the genus Legionella with said nucleic acid molecules are different others. A biochip according to any one of claims 1 to 3, wherein said nucleic acid molecule, where appropriate each of said nucleic acid molecules, is an oligonucleotide obtained by chemical synthesis, preferably about 25 microns in length. at 30 nucleotides. 5. A biochip according to claim 1, comprising, immobilized on said support: at least one rpoB-specific nucleic acid molecule chosen from the nucleic acid molecules of sequences SEQ ID NO: 1 to SEQ ID No. 7 or, for each of said sequences, any sequence having at least 95% identity with said sequence or with its complementary sequence, a sequence comprising it having not more than 25 more nucleotides or a fragment of at least at least 22 consecutive nucleotides included in said sequence, preferably a plurality, preferably all, of said molecules, preferentially at least one mipspecific nucleic acid molecule chosen from the nucleic acid molecules of respective sequences SEQ ID NO: 8 at SEQ ID No. 17 or, for each of said sequences, any sequence having at least 95% identity with said sequence or with its sequence elementary, a sequence comprising it comprising not more than 25 more nucleotides or a fragment of at least 22 consecutive nucleotides included in said sequence, preferably a plurality, preferably all, of said molecules. A biochip according to any one of claims 1 to 5, characterized in that said nucleic acid molecule, where appropriate each of said nucleic acid molecules, is immobilized on said support by means of phosphorus dendrimers having a central nucleus containing at least two functional groups and having at their periphery a first function for their covalent attachment to said support and a second function for the covalent attachment of said nucleic acid molecule. 7. A biochip according to claim 6, wherein the dendrimers are chosen from those consisting of: a central layer in the form of a central core Po, optionally phosphorus, comprising from 2 to 12 functionalized groups, of n layers; intermediate, identical or different, each of said intermediate layers consisting of P1 units corresponding to the following formula (I): -PMFE 15 in which: L is an oxygen, phosphorus, sulfur or nitrogen atom, M represents one of the following groups: an aromatic group di-, tri- or tetrasubstituted with alkyl groups, alkoxy groups, unsaturated groups of the C 1 -C 12 olefinic, azo and acetylenic type, all these groups being able to incorporate or not phosphorus, oxygen, nitrogen, sulfur or halogen atoms, or - an alkyl or alkoxy group having more than one substituent as defined when M is an aromatic group, R1 and R2, which may be may be the same or different, represent a hydrogen atom or one of the following groups: alkyl, alkoxy, aryl, whether or not containing phosphorus, oxygen, sulfur, nitrogen or halogens with R2 being most often different from R1, n is an integer between 1 and 11, E is an oxygen, sulfur or nitrogen atom, said nitrogen atom possibly being bonded to an alkyl, alkoxy or aryl group, all these groups may or may not incorporate phosphorus, oxygen, nitrogen, sulfur or halogen atoms, an outer layer consisting of identical or different units P2, and corresponding to formula (II) in which: W represents one of the following groups: alkyl, alkoxy, aryl, all these groups whether or not containing phosphorus, oxygen, nitrogen, sulfur or halogen atoms, X represents a grouping aldehyde, thiol, amine, epoxide, carboxylic acid, alcohol or phenol. Kit for the detection and / or identification of bacteria of at least one species of the genus Legionella contained in a sample, comprising: a biochip according to any one of claims 1 to 7, a first pair of nucleic primers, optionally labeled, capable of amplifying the sequence of a region of the rpoB gene of said bacteria comprising the portion with the sequence of which the rpoB-specific nucleic acid molecule is able to hybridize specifically, where appropriate each portion with the sequence of which at least one of said rpoB-specific nucleic acid molecules is capable of hybridizing specifically. 9. Kit according to claim 8, wherein said first pair of nucleic primers consists of primers A1 and A2, sequences of SEQ ID No. 18 and SEQ ID No. 19 respectively, or any pair of primers whose sequences. at least 90% identity with said sequences SEQ ID No. 18 and SEQ ID No. 19 or with their complementary reverse sequences. 10. Kit according to any one of claims 8 to 9, further comprising a second pair of optionally labeled nucleic primers capable of amplifying the sequence of a region of the mip gene of said bacteria comprising the portion with the sequence of wherein said mip-specific nucleic acid molecule is capable of specifically hybridizing, where appropriate each portion with the sequence of which at least one of said mip-specific nucleic acid molecules is capable of specifically hybridizing. The kit according to claim 10, wherein said second pair of nucleic primers consists of primers A3 and A4, sequences of SEQ ID No. 20 and SEQ ID No. 21 respectively, or of any pair of primers whose sequences respective ones have at least 90% identity with said sequences SEQ ID No. 20 and SEQ ID No. 21 or with their complementary reverse sequences. 12. A method for detecting and / or in vitro identifying bacteria of at least one species of the genus Legionella contained in a sample, by means of a biochip according to any one of claims 1 to 7, said method comprising: The amplification of a sequence of a region of the rpoB gene of said bacteria comprising the portion with the sequence of which said rpoB-specific nucleic acid molecule is capable of hybridizing specifically, as the case may be each portion with the sequence of which at least one of said rpoB-specific nucleic acid molecules is capable of hybridizing specifically, by contacting with a first pair of specific, optionally labeled nucleic primers, preferably by reaction of polymerization chain, - bringing the amplified DNA into contact with said biochip, and - the detection of a possible hybridization complex. 13. The method according to claim 12, wherein said amplification of a sequence of a region of the rpoB gene of said bacteria is carried out by means of a pair of nucleic primers consisting of primers A1 and A2, respectively SEQ ID sequences. No. 18 and SEQ ID No. 19, or any pair of primers whose respective sequences have at least 90% identity with said sequences SEQ ID No. 18 and SEQ ID No. 19 or with their complementary reverse sequences. The method of any one of claims 12 to 13, comprising: amplifying a sequence of a region of the mip gene of said bacteria comprising the portion with the sequence of which said nucleic acid molecule mip- Specifically, it is capable of hybridizing specifically, where appropriate each portion with the sequence of which at least one of said mip-specific nucleic acid molecules is capable of hybridizing specifically, by contacting with a second pair of nuclei. specific nucleic primers, optionally labeled, preferably by polymerase chain reaction, - bringing the amplified DNA into contact with a biochip according to any one of claims 2 to 7, and - detecting a possible complex of d 'hybridization. 15. The method according to claim 14, wherein said amplification of a sequence of a region of the mip gene of said bacteria is carried out by means of a pair of nucleic primers consisting of primers A3 and A4, respectively SEQ ID N sequences. 20 and SEQ ID No. 21, or any pair of primers whose respective sequences have at least 90% identity with said sequences SEQ ID No. 20 and SEQ ID No. 21 or with their complementary reverse sequences. The method according to any one of claims 14 to 15, wherein the amplification of said sequence of a region of the rpoB gene and the amplification of said sequence of a region of the mip gene of said bacteria are carried out by a method multiplexed chain polymerization reaction system in which the first pair of primers and the second pair of primers are implemented simultaneously. 17. Method according to any one of claims 12 to 16, implemented on a population of nucleic acids previously extracted from the bacteria contained in said sample. 18. Use of a biochip according to any one of claims 1 to 7 for the detection and / or identification of bacteria of at least one species of the genus Legionella, where appropriate a plurality of different species of the genus Legionella. 19. A set of nucleic primers comprising the pairs of primers: primers A1 and A2, with sequences of SEQ ID No. 18 and SEQ ID No. 19, respectively, or any pair of primers whose respective sequences have at least 90 % of identity with said sequences SEQ ID No. 18 and SEQ ID No. 19 or with their complementary reverse sequences, - primers A3 and A4, of sequences SEQ ID No. 20 and SEQ ID No. 21, respectively, or any pair of primers whose respective sequences have at least 90% identity with said sequences SEQ ID No. 20 and SEQ ID No. 21 or with their complementary reverse sequences, at least one primer of each of said pairs being preferentially marked.