WO2018041747A1 - Method for the in vitro detection and identification of at least one target pathogen present in a biological sample - Google Patents

Abstract

The invention relates to a method for the in vitro detection and identification of at least one target pathogen present in a biological sample, implementing a method of gene capturing coupled with high-throughput sequencing, the gene-capturing method allowing the capture of DNA fragments having a length of at least 6000 pairs of bases.

Description

 METHOD OF IN VITRO DETECTION AND IDENTIFICATION OF ONE OR MORE TARGET PATHOGENS PRESENT IN A BIOLOGICAL SAMPLE

TECHNICAL FIELD The present invention relates to a high-throughput method of detection and in vitro identification of one or more target pathogens present in a biological sample.

BACKGROUND

Since the identification of microorganisms in the nineteenth century as one of the major sources of morbidity and mortality, efforts have been made to monitor and control the spread of infectious diseases. In the twentieth century, the advent of antibiotics, mass vaccination and antiviral treatments offered an unprecedented level of control over the spread of these diseases. Nevertheless, infectious diseases remain one of the leading causes of death in the world, with an endless succession of "new" microbial killers emerging every year. Currently available systems for detecting the presence of microorganisms in a sample taken from a host (eg a patient or an animal) are low flow systems. Generally only one microorganism is tested for each sample, unless medical evidence indicates that the host may be suffering from several microbial diseases. This means that only one infection can be detected per test, which has a significant impact on the price and the test time. In addition, in an asymptomatic host, it is often difficult to decide for which microorganism the patient should be tested. Negative results for two or three infectious organisms can give a false sense of security. Cultural approaches have a number of limitations including different culture media suitable for the isolation of various microorganisms, or even the absence of isolation medium. In addition, for certain microorganisms, the growth phases can be tedious leading to isolation steps of several days. Modern detection systems use DNA-based tests to detect infection with pathogenic microorganisms. The most common methods are based on the polymerase chain reaction (PCR) applied directly after nucleic acid extraction or following isolation or enrichment of microorganisms. Thus, these molecular and cultural approaches make it difficult to simultaneously detect several pathogens using a single sample taken from the host organism. These reliable identification problems remain for other approaches, such as DNA microarrays, direct sequencing, and diagnostic approaches, such as immunoassays, microscopic observations, or mass spectrometry.

Therefore, it appears necessary to have a reliable, automatable and inexpensive method allowing the detection and in vitro identification of one or more target pathogens present in a biological sample, even if this or these target pathogens are present in low amounts in the biological sample.

International application WO2015 / 105993 (AgBiome Inc) describes a method for identifying genes of interest implementing a step of gene capture by hybridization followed by a sequencing step to identify said genes of interest. However, the method described in International Application WO2015 / 105993 requires the use of overlapping probes complementary to reference gene sequences to capture DNA fragments and is limited to the capture of short DNA fragments having a maximum length of 1800 base pairs. In addition, the approach does not necessarily allow to identify complete genes let alone nucleic sequences comprising several genes, or even new genes or complete genomes. The method described in international application WO2015 / 105993 only makes it possible to identify genes whose nucleic sequence is close to the targeted genes, and does not make it possible to identify divergent genes, or even new genes. Silke Grumaz et al. (Silke Grumaz et al., Genome Medicine, 2016, Vol 8, pages 1-13) describes a method for detecting pathogens in a plasma sample from skepticemia patients using high throughput sequencing. The method described in Silke Grumaz et al. is to isolate the DNA from the sample using the Qiagen Circulating Nucleic Acid Kit, then sequence and analyze the results using bioinformatic methods. However, the method described in Silke Grumaz et al. is not to enrich the DNA of pathogens from complex biological samples. It does not include steps of preparation of DNA fragments or capture by hybridization. Thus, the pathogen DNA only corresponds to a very small proportion of the sequences obtained (93 to 99% of the sequences correspond to human DNA). It is then difficult or impossible, depending on the abundance to identify complete genes, let alone nucleic sequences comprising several genes, or even new genes or complete genomes.

The patent application US2014 / 0274741 (The Translational Genomics Research) relates to a method for diagnosing neuromuscular diseases in humans, including the detection of one or more mutations in SCML2, CHRND, IFD1, DYNC1 H1, COL6A3, EMD, ARHGAP4, FLNA, MID1 IP1, MID1 and CFP genes. The diagnostic method presented consists of isolating the genomic DNA from the biological sample, hybridizing it with a set of target-specific probes obtained from reference nucleic sequences, purifying the targeted DNA with affinity chromatography, then fragment the DNA obtained to allow its sequencing approaches ^2nd generation. The set of probes used targets at regular intervals reference sequences of 500 to 5000 base pairs, preferably about 2000 base pairs. Thus, large known DNA regions can be enriched from DNA obtained from isolated organisms. However, the method does not make it possible to enrich large regions of unknown DNA beyond the regions covered by the probes. In addition, this method first requires a first depletion step to eliminate by hybridization using probes variants such as pseudogenes, to then specifically enrich during a second hybridization step the genes of the genome. 'interest. Thus, the variants of the target genes are eliminated. The method therefore does not make it possible to identify all the diversity of the genes of interest.

Therefore, it appears necessary to have a high throughput method of detection and identification in vitro more resolvable of one or more target pathogens present in a biological sample, to identify both known and unknown variants genes of interest belonging to the target pathogens responsible for infectious diseases in a host organism, but also to detect new pathogens carrying targeted virulence genes and thus accurately determine the absence or presence of the target pathogen or pathogens present in a given biological sample.

DETAILED DESCRIPTION The applicants therefore propose a new method for detecting and identifying in vitro one or more target pathogens present in a biological sample.

This method, while being more sensitive than existing methods, makes it possible to identify both known and unknown variants of the genes of interest belonging to the target pathogens responsible for infectious diseases in a host organism, but also to detect new pathogens. carriers of targeted virulence genes and thus accurately determine the absence or presence of the target pathogen or pathogens in a given biological sample. An object of the invention is therefore a method for detecting and identifying in vitro one or more target pathogens present in a biological sample, the method comprising the following steps: a) the preparation of a mixture of probes capable of specifically targeting a set of gene sequences of interest belonging to the target pathogens, b) preparing a library of DNA fragments from the biological sample, said DNA fragments contained in the library having a length of at least 6,000 base pairs, c) hybridizing the DNA fragments obtained in step b) by contacting said DNA fragments with the probe mixture of step a), d) the capture of the hybridization complexes of step c), e) the sequencing of the DNA fragments captured in step d), f) the bioinformatic reconstruction of the genes of interest and / or the genome of the microorganism (s). organisms from captured DNA fragments sequenced from step e), and g) identifying the target pathogen (s) present in the biological sample.

The method according to the invention makes it possible to capture DNA fragments having a length of at least 6,000 base pairs. With this new method, the inventors are able to identify both known and unknown sequences of interest genes belonging to target pathogens responsible for infectious diseases in a host organism. In addition, the inventors using this method reveal genes present in the regions adjacent to the target genes making it possible to reveal new pathogenic strains and thus to accurately determine the absence or the presence of the target pathogen (s) present in the region. a given biological sample. This method, while making it possible to exhaustively explore the genetic diversity of genes of interest, has the advantage of ensuring the identification of the flanking regions associated with the targeted sequences. It is then possible, through the characterization of large DNA regions, to highlight known or unknown genomic organizations and thus to identify genes that may have a role in the development, survival and adaptation of pathogens. It is also possible to reconstruct the genomes carrying the targeted sequences that can contribute to the improvement of the diagnosis, but also to provide information that can facilitate the isolation of these pathogens, while highlighting potential new therapeutic targets. Thus, by reconstructing large flanking regions with targeted genes and simultaneous detection of multiple sequences, pathogen identification is possible down to the level of the species, or even the strain or individual, representing the level. most resolutive identification. Finally, the present method makes it possible to dispense with the use of overlapping probes and complementary reference sequences. As a result, it is not necessary to have an exhaustive initial knowledge of the complete sequence of genes to be captured. The approach makes it possible to reveal unknown genes thanks to the exploratory character of the probes resulting in their degeneration at particular positions.

The method according to the invention is capable of detecting and identifying one or more target pathogens present in a biological sample. By "target pathogens" is meant any infectious biological agent capable of inducing infectious disease in a host organism.

In an advantageous embodiment of the invention, the target pathogens can be all types of infectious agents. In a particularly advantageous embodiment, the target pathogens may be chosen from the group comprising: bacteria, archaea, viruses, viroids, fungi, in particular yeasts, unicellular eukaryotes (conventionally called protists), in particular the protozoa and worms. In an advantageous embodiment of the invention, the target pathogens may be bacteria. In an advantageous embodiment of the invention, the bacteria may be monoderm-type walled bacteria (classically called Gram-positive), of the diderme type (typically called Gram-negative), or showing particular resistances such as AFB (Bacteria). Acido Alcoholo Resistant) like mycobacteria or even wall-free bacteria like mycoplasma.

In a particularly advantageous embodiment of the invention, the target bacteria may be chosen from the group comprising: Bacillus anthracis, Clostridium botulinum, Francisella tularensis, Yersinia pestis, Brucella, Burkholderia, Campylobacter jejuni, Chlamydiophila psittaci, Coxiella burnetii, Escherichia coli , Clostridium perfringens, Listeria monocytogenes, Vibrio, Salmonella, Shigella, Staphylococcus aureus, Rickettsia, Yersinia enterocolitica, Mycobacterium tuberculosis, Anaplasma phagocytophilum, Bartonella henselae, Bordetella pertussis, Borrelia miyamotoi, Clostridium difficile, Ehrlichia, Enterococcus, Leptospira, Borrelia burgdorferi, Streptococcus pyogenes, Legionella, Mycoplasma, Nocardia, Pneumocystis, Candida, or a mixture of two or more of these target bacteria.

"Biological sample" means any sample originating from a host organism having at least nucleic acids of another species, the nucleic acids of the other species possibly being nucleic acids of the DNA or RNA type that can be converted into Complementary DNA (cDNA), such as, for example, viral DNA or RNA, bacterial DNA or RNA, archaic DNA or RNA, fungal DNA or RNA, such as yeast DNA or RNA, RNA viroid, eukaryotic unicellular DNA or RNA, worm DNA or RNA, or protozoan DNA or RNA. In an advantageous embodiment of the invention, the biological sample can be obtained from a body fluid or a tissue taken from the animal or from humans or from the plant.

In an even more advantageous embodiment of the invention, the biological sample may be selected from the group consisting of: a whole blood sample, a plasma sample, a serum sample, a saliva sample, a secretion sample nasal, a urine sample, a stool sample, a cerebrospinal fluid sample, a sperm sample, a vaginal secretion sample, and a biopsy specimen.

The method developed by the inventors does not require prior samples purified pathogens, but is capable of identifying homologous sequences both from isolated organisms, and directly from uncharacterized mixtures of populations. organisms comprising the targeted pathogen (s) taken directly from raw biological samples and not subjected to purification. In this way, the method according to the invention is capable of identifying gene sequences and variants thereof, from pathogens that are not cultivable or difficult to cultivate, but also poorly represented within the sample studied.

Genes of interest

Known DNA sequences of genes of interest or variants thereof, but also unknown sequences of genes of interest or variants thereof can be detected and identified by the method according to the invention. Within the meaning of the present invention, the term "gene sequence of gene of interest" is understood to mean the known or unknown gene DNA sequence. In one embodiment according to the invention, the sequences of the genes of interest can be sequences corresponding to the virulence genes of the pathogens, to the antibiotic resistance genes, to the regulatory genes to functional markers, or to sequences repeated.

In another embodiment of the invention, the probes may be designed to recognize 16S rDNA sequences, or any other phylogenetic differential sequence such as mcrA, col genes or STIs. In this way, the method according to the invention is capable of capturing sufficient portions of the 16S rDNA or complete 16S rDNAs, or sufficient or complete portions of the other phylogenetic markers, needed to estimate the distribution and the identification of the taxa present in the biological sample.

Table 1 below gives examples of pathogens and target genes that can be detected and identified in the method according to the invention. It is important to note that these pathogens and target genes are provided only as examples. They do not represent an exhaustive list of pathogens and genes of interest that can be targeted by this method.

Pathogens Target genes

 Bacillus anthracis acpB, atxA, bsIA, capA, capB, capC, capD, cya, lef, pagA, rpoB, sap, vrrA, vrrB

Clostridium botulinum bontA, bonB, bonC, bonD, bonE, bonF, bonG, flAa, flaB, ntnH, sigE, sigG, sigK, spoOA

Francisella tularensis acpA, asd, capB, capC, fopA, fsaP, galE, iglC, pdpD, pepO, tu 14

Yersinia pestis garlic, coffee1, glpD, hmsF, hmsH, hmsR, IcrV, pla, rpoB, yihN, ymt, yopT

Brucella alkB, BCSP31, BMEI1162, bp26, IS711, omp25, omp31, rpsL, wobA

Burkholderia bimA, fliC, fliP, motB, mprA

Campylobacter jejuni cadF, cdtA, cdtB, cdtC, ceuB, ceuC, ceuD, cEe, ciaB, ciaC, hipO, mapA Chlamydiophila psittaci enoA, envB, incA, ompA

Coxiella burnetii adaA, cbbE, cbhE, coml, htpB, icd

Escherichia coli aggR, astA, bfpA, bfpB, cadA, eaeA, ehxA, eltA, eltB, escV, hlyA, rfbE, saa, sta, stxlA, stxlB, stx2A, stx2B, vt1

Clostridium perfringens cpa, cpb, cpb2, cpe, etx, iap, netB, tpeL

Listeria monocytogenes actA, aut, bsh, fbpA, gtcA, hlyA, iap, inlA, inlB, mpl, oatA, pgdA, plcA, plcB, prfA, svpA, vip

Vibrio ace, ctxA, ctxB, hlyA, tlh, trh, vvhA, zot

Salmonella cdtB, invA, misL, rck, sipB, sipC, spvC, stn

Shigella ial, ipaB, ipaH, setlA, setlB, stxA, stxB, virA

Staphylococcus aureus coa, etaA, naked, sea, seb, sec, seg, sei, sej, salt, tst

Ric etisia prowazekii gtlA, ompB

Yersinia enterocolitica garlic, blaA, foxA, myf, naked, rfbB, virF, wbbU, wbcA, yadA, ystA, ystB

Rickettsia gtlA, ompA, ompB

Mycobacterium tuberculosis katG, gyrB, erp, hspX, lepB, mtp40, pncA, embB, gyrA

Anaplasma ank, msp2, msp4

Bartonella henselae BadA, GltA, GroEL, htrA, pap31

Bordetella pertussis bapC, cyaA, dnt, prn, ptxA, tcfA

Borrelia miyamotoi flaB, glpQ, ospA, recG

Clostridium difficile cdtA, cdtB, gdh, tcdA, tcdB, tcdC

Ehrlichia dsb, groEL, map1, rpoB, sodB Enterococcus faecium ace, asal, cylA, ddl, efaA, esp, freeze, hyl

Leptospira gyrB, Ifb1, lig, HpL32, ompL1, secY

Borrelia burgdorferi ospA, ospB, p66

Streptococcus pyogenes sagA, slo, smeZ, spec, speg, speh, spei, spej, spem, ssa

Table 1: Example of pathogens and target genes.

The method according to the invention can identify variants of known or unknown sequences from several families of genes of interest. For the purposes of the present invention, the term "variants" or "variant gene" refers to genes showing more or less important sequence similarities. Although the expression of a variant may be modified with respect to that of the gene of interest, the variant may retain the same function as the gene of interest or lead to new functions. For example, a variant may have increased expression, reduced expression, a different expression spectrum (for example, for an insecticidal toxin gene), or any other modification of the expression relative to the targeted gene of interest. In general, by "variant" is meant substantially similar sequences. For polynucleotides, a variant comprises deletion and / or insertion of one or more nucleotides at one or more sites in the native polynucleotide, or substitution of one or more nucleotides at one or more sites in the native polynucleotide or even chemical methylation or other modifications. By "native polynucleotide" or "wild-type polynucleotide" is meant a polynucleotide which comprises a natural nucleotide sequence. For protein coding sequences, the variants may be so-called "conservative" variants which, because of the degeneracy of the genetic code, encode the native amino acid sequence of the product of the gene of interest; natural allelic variants, such as those which can be identified by techniques well known to those skilled in the art, such as, for example, by polymerase chain reaction amplification (PCR) or hybridization or direct sequencing techniques without priori; artificially obtained variants, such as those generated by random or directed mutagenesis but still coding for the gene product of interest or showing sequence and structure similarity. Advantageously, the variant has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence of the gene of interest. probes

In one embodiment of the invention, the method utilizes capture probes specific for the genes of interest or known or unknown variants thereof. Within the meaning of the present invention, the term "probe" is intended to mean a polynucleotide capable of hybridizing with the sequence of the gene of interest or a known or unknown variant thereof. The probes can be RNA or DNA sequences, overlapping, contiguous or sequential. In an advantageous embodiment of the invention, the probes are single-stranded RNA sequences capable of hybridizing to the sequence of the gene of interest by complementarity of the nucleotides. By way of example, the RNA sequence of the probe is complementary to the DNA, cDNA or RNA sequence of the fragment of the gene of interest. In a particularly advantageous embodiment, the probes according to the invention are non-overlapping single-stranded RNA sequences. In an advantageous embodiment, the probes are exploratory, that is to say capable of specifically targeting a set of known and unknown sequences of genes of interest, the known and unknown sequences of genes of interest being sequences DNA or RNA or cDNA. In an advantageous embodiment according to the invention, the probes are non-overlapping and exploratory single-stranded RNA sequences.

In an advantageous embodiment according to the invention, the probes have a length of at least 20 contiguous nucleotides, advantageously at least 30 contiguous nucleotides, advantageously at least 40 contiguous nucleotides, advantageously at least 50 contiguous nucleotides , advantageously at least 60 contiguous nucleotides, advantageously at least 70 contiguous nucleotides, advantageously at least 80 contiguous nucleotides, advantageously at least 90 contiguous nucleotides, advantageously at least 100 contiguous nucleotides, advantageously at least 1 10 contiguous nucleotides, advantageously at least 120 contiguous nucleotides, advantageously at least 130 contiguous nucleotides, advantageously at least 140 contiguous nucleotides, advantageously at least 150 contiguous nucleotides or more. Advantageously, the sequence of the probes may comprise from 20 to 150 contiguous nucleotides, advantageously from 70 to 150 contiguous nucleotides, advantageously from 100 to 140 contiguous nucleotides, advantageously from 1 to 120 contiguous nucleotides, advantageously from 70 to 90 contiguous nucleotides. Particularly advantageously, the probes have a length of 80 contiguous nucleotides.

In an advantageous embodiment of the invention, the interval between two probes is between 10 base pairs (bp) and 1000 bp, advantageously between 50 bp and 500 bp, advantageously between 100 bp and 450 bp, advantageously between 100 bp and 400 bp, advantageously between 100 bp and 350 bp, advantageously between 100 and 300 bp, advantageously between 100 bp and 250 bp, advantageously between 100 bp and 200 bp. In a particularly advantageous embodiment of the invention, the interval between two probes is between 100 bp and 200 bp.

In an advantageous embodiment according to the invention, the probes can be complemented with a detectable marker to allow capture of the hybridization complex comprising the probe hybridized to the DNA or RNA fragment or cDNA of the gene of the invention. interest or known or unknown variant of it. In a particularly advantageous embodiment, the probes are labeled with biotin, a hapten or an affinity tag, or are generated by polymerization or transcription using oligonucleotides. In an even more advantageous embodiment, the RNA probes are biotinylated. In an even more advantageous embodiment, the probes are biotinylated single-stranded RNA probes having a length of 80 nucleotides. In an even more advantageous embodiment, the single-stranded RNA probes are biotinylated randomly over their entire sequence. Oligonucleotide probes may include adapters necessary for their PCR amplification. In a particularly advantageous embodiment, the probes comprise an adapter A of sequence SEQ ID No. 1 and an adapter B of sequence SEQ ID No. 2, the adapter A being placed at the 5 'end of the sequence of the probe and the adapter B being placed at the end 3 'of the sequence of the probe. Advantageously, the sequences of the adapters A (SEQ ID No.1), and B (SEQ ID No.2) are added to each end of the probes leading to probes whose sequence is of the "adapter A-adapter-B adapter" type. . The probe may also include a promoter, in position 5 'of the adapter A, or in 3' of the adapter B, for example the T7 promoter, of sequence SEQ ID No.3.

In one embodiment of the invention, the oligonucleotide probes, once synthesized, are amplified using adapters A and B to add the T7 promoter, and then purified. Those skilled in the art will know how to use all the appropriate methods for amplification and purification. Advantageously, the probes can be amplified using the Platinium Taq DNA Polymerase High Fidelity kit marketed by Invitrogen and can be purified using the MinElute PCR purification kit marketed by Qiagen. The probes are then transcribed in vitro using the T7 promoter in the presence of biotinylated dNTPs to obtain the biotinylated RNA probes. Those skilled in the art will know how to use all the appropriate methods for transcribing the probes bioyinylated and purify them. Advantageously, the in vitro transcription can be carried out using the MEGAScript kit sold by the company Ambion and biotinylated dUTPs marketed by the company Tebu-Bio. The biotinylated RNA probes can then be purified using the RNeasy kit sold by the company Qiagen. In one embodiment of the invention, probes specific for different genes and / or pathogens can be used simultaneously to hybridize with DNA or RNA or cDNA fragments prepared from the sample. organic. For example, for the analysis of a biological sample, probes designed for each gene of interest can be combined to form a probe mixture, upstream or at the time of contact of the probes with the fragment library. DNA obtained from the biological sample.

Within the meaning of the present invention, a "probe mixture" refers to a set of probes designed to be specific for one or more genes of interest and its known or unknown variants belonging to a particular target pathogen and / or set of probes designed to be specific for one or more genes of interest belonging to different target pathogens.

In a particularly advantageous embodiment of the invention, the probe mixture may comprise at least one probe, advantageously at least two different probes, advantageously at least 10 different probes, advantageously at least 20 different probes, advantageously at least 30 probes. different, advantageously at least 40 different probes, preferably at least 50 different probes, preferably at least 60 different probes, preferably at least 70 different probes, preferably at least 80 different probes, preferably at least 90 different probes, preferably at least 100 different probes preferably at least 200 different probes, advantageously at least 300 different probes, advantageously at least 400 different probes, advantageously at least 500 different probes, advantageously at least 600 different probes, advantageously at least 700 different probes, advantageously at least 800 different probes, advantageously at least 900 different probes, advantageously at least 1000 different probes, advantageously at least 1200 different probes, advantageously at least 1400 different probes, advantageously at least 1500 different probes, advantageously at least 1600 different probes, advantageously at least 1700 probes different, advantageously at least 1800 different probes, preferably at least 1900 different probes, advantageously at least 2000 different probes, advantageously at least 3000 different probes, advantageously at least 5000 different probes or more.

In a particularly advantageous embodiment of the invention, the mixture of probes comprises between 1 and 5000 different probes, advantageously between 100 and 4500 different probes, advantageously between 500 and 4000 different probes, advantageously between 1000 and 4000 different probes, advantageously between 1500 and 3000 different probes, advantageously between 1600 and 2500 different probes, advantageously between 1900 and 2100 different probes.

In a particularly advantageous embodiment of the invention, the probe mixture may be specific of at least 1, advantageously at least 2, advantageously at least 10, advantageously at least 50, advantageously at least 100, advantageously at least 150, preferably at least 200, advantageously at least 250, advantageously at least 300, advantageously at least 500, at least 750, advantageously at least 800, advantageously at least 900, advantageously at least 1000, or any other number of genes of interest. Advantageously, the probe mixture is specific for at least 100 genes of interest. Advantageously, the probe mixture is specific for at least 200 genes of interest.

In a particularly advantageous embodiment of the invention, the number of genes of interest targeted by the probe mixture is between 1 and 1000, advantageously between 10 and 500 genes of interest, advantageously between 50 and 4500 genes. interest, advantageously between 100 and 400 genes of interest, preferably between 200 and 300 genes of interest.

Within the meaning of the present invention, a probe specific for a gene and all of its known or unknown variants is designed to hybridize all the genes and known or unknown variants of this gene present in the sample. organic. A probe is therefore specific for all the variants of a gene of interest. In an advantageous embodiment, the sequences of the probes are designed to target the genes of interest and their known or unknown variants using software tools such as KASpOD, HiSpOD or ExSpOD. In an advantageous embodiment according to the invention, the probe is capable of hybridizing with a fragment of DNA or RNA or cDNA of the gene of interest and its known or unknown variants of a length of d at least 6,000 nucleotides, advantageously 20,000 nucleotides more preferably 50,000 nucleotides, up to the total length of the nucleotide sequence of the genome of interest.

Preparation of a library of DNA fragments from the biological sample To obtain the library of DNA fragments capable of being hybridized by the probes as described above, the biological sample containing the pathogenic DNAs must be prepared for the hybridization step. Extraction of the DNA from the biological sample can be carried out by any technique well known to those skilled in the art, making it possible to extract DNA of size compatible with the size of the fragments to be captured. for the hybridization step. Advantageously, the extraction of the DNA from the biological sample can be carried out using commercially available DNA extraction kits, for example the QIAamp DNA Blood or DNeasy Blood & Tissue kits marketed by QIAGEN. , and UltraClean Tissue & Cells or UltraClean BloodSpin marketed by MoBio. In an advantageous embodiment of the invention, the extracted DNA is then fragmented into DNA fragments of specific length. Advantageously, the DNA is fragmented into fragments having a length of at least 6,000 base pairs, advantageously at least 20,000 base pairs, advantageously at least 50,000 base pairs. The fragmentation of the DNA can be carried out by any technique known to those skilled in the art to obtain DNA fragments having the desired length. Advantageously, the DNA fragments according to the invention are obtained using the g-TUBE kit marketed by Covaris.

In a particularly advantageous embodiment of the invention, the DNA fragments have either a length of 6000 base pairs, a length of 20 000 base pairs of DNA, or a length of 50 000 base pairs. DNA. For the purposes of the present invention, "DNA fragment library" or "library" means a set of DNA fragments having the same length, said DNA fragments being obtained from the biological sample. . In an advantageous embodiment of the invention, the DNA fragments constituting the library have a homogeneous size. In a particular embodiment of the invention, three libraries of DNA fragments can be obtained from the biological sample: a so-called "6kb" library comprising DNA fragments having an average length of 6,000 pairs of DNAs. bases, a so-called "20kb" library comprising DNA fragments having an average length of 20,000 pairs and a so-called "50kb" library comprising DNA fragments having an average length of 50,000 base pairs. In a particularly advantageous embodiment of the invention, the library of DNA fragments comprises DNA fragments having a length of at least 6,000 base pairs, advantageously at least 20,000 base pairs, advantageously at least 50,000 base pairs.

In an advantageous embodiment, the method according to the invention may further comprise a size selection step of the fragmented DNAs. Advantageously, the selection step can be carried out with the BluePippin system using the 0.75% Agarose Gel Cassette Mid Range Targets kits, 0.75% Agarose Gel Cassette Low Range Targets or 0.75% Agarose Gel Cassette 50kb marketed by the company SageScience.

Advantageously, this selection step makes it possible to retain only the DNA fragments having an average length of 6,000 base pairs, an average length of 20,000 base pairs, or an average length of 50,000 base pairs. DNA. In an advantageous embodiment of the invention, the libraries of DNA fragments can be enriched by amplification, in particular by isothermal amplification by strand displacement, making it possible to enrich at least 1.5 times, advantageously at least 2 times. advantageously at least 5 times, advantageously at least 10 times, advantageously at least 20 times, advantageously at least 50 times, advantageously at least 100 times, advantageously at least 1000 times the target population of fragmented DNA. Advantageously, the amplification of the library can be carried out with the IHustraGenomPhi V2 DNA Amplification kit marketed by GE Healthcare.

In an advantageous embodiment, the method according to the invention may further, after the amplification step, comprise a step of selecting the size of the libraries of amplified DNA fragments. Advantageously, the step of selecting the size of the libraries of amplified DNA fragments can be carried out with the BluePippin system using the 0.75% Agarose Gel Cassette Mid Range Targets Kit, 0.75% Agarose Gel Cassette Low Range Targets or 0.75% Agarose Gel Cassette 50kb marketed by the company SageScience. Advantageously, this step of selecting the size of the libraries of amplified DNA fragments makes it possible to preserve only the amplified DNA fragments having an average length of 6,000 base pairs, or an average length of 20,000 base pairs, an average length of 50,000 base pairs of DNA. Hybridization and capture of DNA fragments

 Hybridization

The probes may be mixed with the DNA fragment library prior to the hybridization step by any means known to those skilled in the art. The amount of probes added to the DNA fragment library must be sufficient to allow hybridization of all DNA fragments carrying the genes of interest or its known or unknown variants. In an advantageous embodiment according to the invention, the number of probes introduced into the mixture is greater than or equal to the number of DNA fragments contained in the bank. The probe / DNA fragment ratio for hybridization is about 1: 1, preferably about 250: 1, preferably about 1000: 1, preferably about 20,000: 1 or greater.

In an advantageous embodiment according to the invention, the hybridization step c) is carried out in solution or on a solid support. Advantageously, the hybridization step is carried out in solution. In an advantageous embodiment according to the invention, the hybridization step is carried out under stringent conditions.

In a particular embodiment, the library of DNA, cDNA or RNA fragments is hybridized with the probes for a duration of at least 16 hours, advantageously at least 20 hours, even more advantageously of at least 24 hours, and at a temperature of at least 45 ° C, preferably at least 50 ° C, preferably at least 55 ° C, preferably at least 60 ° C, still more preferably at least minus 65 ° C. In a particularly advantageous embodiment, the DNA fragment library is hybridized with the probes for 24 hours at a temperature of 65 ° C. Advantageously, the hybridization buffer may comprise a buffer based on a mixture of sodium chloride, EDTA and phosphate, sold under the name SSPE ("Saline-Sodium Phosphate-EDTA Hybridization Buffer") buffer, a solution Denhardt's, EDTA, SDS buffer, EDTA and water. Advantageously, the hybridization buffer used in the present invention for the hybridization step comprises 20X SSPE at a concentration of 10X, Denhardt's 50X at a concentration of 10X, 0.5mM EDTA pH 8 at a concentration of 10mM, 10% SDS at a concentration of 0.2% and water.

Capture

After hybridization, the biotinylated probes can be separated according to the presence of the detectable label, and the unbound probes and DNA fragments are removed in appropriate washing conditions. Only those DNA fragments that hybridize specifically with the biotinylated probes are retained. The hybridization complexes can be captured and purified from the mixture of unbound probes and unbound DNA fragments of the library. For example, the hybridization complexes can be captured using a streptavidin molecule attached to a solid phase, such as a magnetic bead. In a particularly advantageous embodiment according to the invention, the hybridization complexes are captured using streptavidin-coated magnetic beads, advantageously using 500 ng of Dynabeads M-280 beads (Life Technologies). In an advantageous embodiment according to the invention, the step d) of capture is followed by three washing steps to eliminate the probes and unbound DNA fragments, these three washing steps using three wash buffers different.

Advantageously, the first washing is carried out using a washing buffer comprising sodium chloride, advantageously at a concentration of between 0.5M and 1.5M, advantageously at a concentration of 1M, of Tris-HCl, advantageously at a concentration of between 5 mM and 15 mM, advantageously at a concentration of 10 mM, and EDTA, advantageously at a concentration of between 0.5 mM and 1.5 mM, advantageously at a concentration of 1 mM. Advantageously, the first washing is carried out at ambient temperature, advantageously at a temperature of between 25 ° C. and 30 ° C. Advantageously, the first washing is carried out at a pH of between 7.0 and 8.0, advantageously at a pH of 7.5.

Advantageously, the second washing is carried out using a second elution buffer comprising 20X SSC (SSC 20X = 3M NaCl, 0.3M sodium citrate), advantageously at a concentration of between 0.5X and 1.5X, advantageously at a concentration of 1 X and 10% SDS, advantageously at a concentration of between 0.05% and 0.15%, advantageously at a concentration of 0.1%. Advantageously, the second washing is carried out at ambient temperature, advantageously at a temperature of between 25 ° C. and 30 ° C.

Advantageously, the third washing is carried out using a third elution buffer comprising 20X SSC, advantageously at a concentration of between 0.05X and 0.15X, advantageously at a concentration of 0.1% and 10% SDS, advantageously at a concentration of between 0.05% and 0.15%, advantageously at a concentration of 0.1%. Advantageously, the third washing is carried out at a temperature of between 30 ° C. and 80 ° C., advantageously at a temperature of between 40 ° C. and 75 ° C. advantageously at a temperature of between 50 ° C. and 70 ° C., advantageously at a temperature of 65 ° C.

In an advantageous embodiment according to the invention, the washing steps may be followed by an elution step by chemical denaturation with sodium hydroxide, making it possible to separate the hybridization complexes and thus release the captured DNA fragments. Advantageously, this step makes it possible to separate the probes from the captured DNA fragments by sedimenting the streptavidin-coated magnetic beads on which the probes are fixed. The captured DNA fragments are then recovered in the supernatant. In an advantageous embodiment, the elution step can be carried out by incubating the streptavidin-coated magnetic beads on which the hybridization complexes are fixed in the presence of sodium hydroxide at a concentration of between 0.01 M and 1 M, advantageously between 0.05M and 0.5M, advantageously 0.1M, for at least 5 minutes, advantageously 10 minutes, at room temperature. The magnetic beads coated with streptavidin sediment and the supernatant is then recovered. A Tris-HCl buffer, advantageously at a concentration of between 0.5M and 1.5M, advantageously at a concentration of 1M, at pH 7.5 is then added to the supernatant.

In an advantageous embodiment according to the invention, the elution step may be followed by a purification step of the captured DNA fragments. Advantageously, the purification step can be carried out with the Microcon DNA fast flow PCR grade kit, centrifugal filters, ETO treated dual cycle, marketed by Merck Millipore.

Amplification of captured DNA fragments

In an advantageous embodiment, the method according to the invention may further comprise a step of amplification of the DNA fragments resulting from step d) capture. Advantageously, the amplification of the library can be carried out with the IHustraGenomPhi V2 DNA Amplification kit marketed by GE Healthcare.

Selection of DNA fragments captured according to their size In an advantageous embodiment, the method according to the invention may further comprise a step of selecting the DNA fragments captured according to their size. resulting from step d) capture. Advantageously, the selection step can be carried out with the BluePippin system using the 0.75% Agarose Gel Cassette Mid Range Targets kit, 0.75% Agarose Gel Cassette Low Range Targets or 0.75% Agarose Gel Cassette 50kb marketed by the company SageScience. Advantageously, this selection step makes it possible to retain only the DNA fragments having an average length of 6,000 base pairs, an average length of 20,000 base pairs, or an average length of 50,000 base pairs. DNA or RNA or cDNA.

The size distribution of the captured DNAs can then be evaluated using the Agilent DNA 12000 kit marketed by Agilent.

Sequencing of captured DNA fragments

The captured DNA fragments can then be sequenced by any technique well known to those skilled in the art. Sequencing of the captured DNA fragments can be performed using the high throughput sequencing systems marketed by Illumina Inc., for example the MiSeq or HiSeq system. All platforms known to those skilled in the art, such as first generation platforms such as Sanger, second generation such as Illumina (MiniSeq, MiSeq, NextSeq, HiSeq, Synthetic Long Read, 10X Genomics) or 454 (Roche) or SOLiD (Thermo Fisher) or Ion Torrent (Thermo Fisher) or Gene Reader (Qiagen) or Complete Genomics (GGI) and third generation such as Pacific BioSciences (RSII or Sequel) or Oxford Nanopore (MinlON, GridION or PromethlON ) as well as new platforms under development (GenapSys, Genia, Firefly, NanoString Technologies, GnuBio, Electron Optica) can be used to sequence captured DNA fragments. Depending on the sequencing method used, those skilled in the art will be able to construct the adapted libraries from the captured DNA fragments.

Reconstruction of large fragments of genomes

The sequences of the different DNA fragments of the genes of interest can be assembled by any means known to those skilled in the art. The sequences of the different DNA fragments of the genes of interest can be assembled to reconstruct the total sequence of the gene of interest, or a known or unknown variant thereof. The sequences of the different DNA fragments of the genes of interest can be assembled to reconstruct the total sequence of the gene of interest, or a known or unknown variant thereof and the flanking regions associated with the targeted sequences or even the entire genome in which is located the gene of interest, thus allowing its identification. In some embodiments, the sequences are assembled using bioinformatic tools, such as IDBA-UD, Spades, MetaVelvet. After assembly, the sequences of the genes of interest, or known or unknown variants thereof, are confirmed by comparison of the sequences against databases of known sequences, such as the GenBank NCBI or ENA database. EMBL, in order to characterize the levels of similarity with the known sequences of the gene of interest, or a variant thereof.

In a particularly advantageous embodiment of the invention, the method for the detection and in vitro identification of one or more target pathogens present in a biological sample comprises the following steps: a) the preparation of a mixture of probes non-overlapping and exploratory methods capable of specifically targeting a set of gene sequences of interest belonging to the target pathogens, b) preparing a library of DNA fragments from the biological sample, said DNA fragments contained in the library having a length of at least 6,000 base pairs, c) hybridization of the DNA fragments obtained in step b) by contacting said DNA fragments with the probe mixture of the step a), d) capturing the hybridization complexes of step c), e) sequencing the DNA fragments captured in step d), and f) bioinformatic reconstruction of the genes of interest and / or of the genome of the micro-gold mechanisms from the captured DNA fragments sequenced from step e), and g) identifying the target pathogen (s) present in the biological sample.

The following examples illustrate the invention. EXAMPLES

Example 1 Identification of pathogens involved in acts of bioterrorism in a biological sample

A / Determination of the probes: A set of 1685 degenerate probes of 80 nucleotides targeting virulence genes or functional genes enabling the identification of priority bacterial pathogens (Table 2) for bioterrorism was determined using KASpOD software (K -mer based Algorithm for High-Specific Oligonucleotide Design (Parisot N. et al., 2012, vol 28, pages 3161-3162) and HiSpOD (High Specifies Oligo Design) (Dugat-Bony E et al., 201 1, vol. 27, 641-648).

A set of 15 probes having a length of 28 to 50 nucleotides was determined to allow enrichment of the 16S rDNA or rRNA of all prokaryotes, including the pathogens listed in Table 2.

B / Results obtained

Through the determination of this set of exploratory capture probes targeting virulence genes or other functional genes of bioterrorism bacterial pathogens and the 16S rRNA genes of all prokaryotes, it is possible to detect and identify accurately all pathogens targeted within any type of biological sample. The probe set can be used in its entirety to provide a comprehensive diagnostic tool for bioterrorism pathogens, or only probes targeting a microorganism or a gene encoding particular functions (eg all genes coding for toxins) can be targeted. to provide more specific diagnostic tools. Thanks to the reconstruction of large genomic regions around biomarkers and the simultaneous detection of several of them for each pathogen, the capture method using this set of probes can allow a simultaneous and resolutive identification of the pathogens present within the biomarker. 'sample. In addition, thanks to the exploratory nature of the probes, gene variants as well as new pathogens can be identified. EXAMPLE 2 Identification and Detection of a Pathogenic Bacteria Within a Microbial Community

A / Preparation of an artificial microbial community:

An artificial mixture of 21 bacterial species and 7 archaeal species (representing 6 phyla, 13 classes, 19 orders, 23 families and 26 genera) whose genomes are sequenced, was made from genomic DNA extracted from pure cultures of different species. (DSMZ) (Table 6). Abundances of different species (based on the number of genomes in the mixture) were defined so that the final community profile reflects species abundance variability in an environmental microbial community.

Table 6: Structure of the Artificial Microbial Community

 Species N ° DSMZ Abundances (%)

Halomicrobium mukohataei 12286 25

 Saccharophagus degradans 17024 25

 Tsukamurella paurometabola 20162 15

 Clostridium acetobutylicum 792 10

Roseobacter denitrificans 7001 5

Novosphingobium pentaromativorans 17173 5

 Geobacter lovleyi 17278 3

Ruegeria Pomeroyi 15171 3

Desulfovibrio vulgaris subsp. vulgaris 644 2

 Pedobacter heparinus 2366 2

Corynebacterium glutamicum

 20300 1

 (target organism)

 Cellulomonas flavigena 20109 1

Pseudomonas putida 6125 1

Methanoculleus marisnigri 1498 0.5

Saccharopolyspora erythraea 40517 0.5

 Halogeometricum borinquense 1 1551 0.3

 Planctomyces limnophilus 3776 0.3

Lactobacillus delbrueckii subsp. bulgaricus 20081 0.2

 Methanospirillum hungateii 864 0.1

Lactobacillus brevis 20054 0.07

Methanocorpusculum labreanum 4855 0.01 Flavobacterium psychrophilum 21280 0.01

 Streptomyces avermitilis 46492 0.006

Listeria welshimeri 20650 0.002

 Sphingobium indicum 16412 0.001

Clostridium leptum 753 0.0008

 Methanobrevibacter smithii 861 0.0001

Methanococcus aeolicus 17508 0.0001

B / Determination and synthesis of the probes:

 The pepO gene, encoding an endopeptidase involved in regulating the expression and secretion of virulence factors of Flavobacterium psychrophilum, the bacterial agent responsible for flavobacteriosis in fish of the salmonid family, was targeted by probes. Thus, a set of 1 1 probes specific for 80 nucleotides targeting the gene of interest was determined, using KASpOD software (K-mer based Algorithm for high-specific oligonucleotide design) (Parisot N et al., 2012, vol. 28, pages 3161-3162) and HiSpOD (High Specifies Oligo Design) (Dugat-Bony E et al., Bioinformatics, 201 1, vol 27, pp. 641-648) (SEQ ID NO: 1704-1714).

Table 7: Sequences of capture probes targeting the pepO gene of Flavobacterium psychrophilum.

 SEQ ID No. Probe Sequences

 TTACGAATAGTTTGTCTCCTTCTTTTATATCAAAAGCTTTGTA

 1704

 AAAAGCATCGAGA I I I GTAAGTGGCACATAGGCTCTG

AATAATCTTTGTTCTGGGGTGTATCCATCTATAAGACCTGGA

 1705

 TTTCCGTTTTGTTTTAAATACAATTGAAGGCCATCGTA

TGTAAATTGTTTTAAATCATCTGCTGTCCACCAGTCTACTAA

 1706

 A I I I CCGTCGGCATTGTATCGTGCTCCAGAATCATCGA

ATGCAGGATTGTAGTAAGCGTTTACTGTTTGTGGCGACATT

 1707

 CCCCATGTTGTTTTGTCGACTGGTTTTTTTAGTTCTGCG

TTGATGGTTACTTTGTGTAGTTTTTCGACAGCCTTAATTTTAG

 1708

 TTTCGACAGACATCCATGTTAAATTATTTATTCTGTT

CAGTGCTTTTTCGTTTCTAGGGCGTTGTTTTTTTGCTCCTTG

 1709

 TAATGTTTTGCTGTAAAATTCCCAGTTTGCGGTTTCTA

AAACTACAATTGATTGTAAATTAGTAATCCCAACGGCATCTA

 1710

 AATAATTTTTCCAGTGTATAGATGGTGTTAGTTTTTGT

TCTG CTG GTTTTGTTCCTAAAAACTGTAG CATTCTCG CTACA

 171 1

 TGAAGTACATATTTCTCACGTTTTTCTTTCGAATCCTT

GCCAAGAGGTTCCATTTCTATTATTAGATTGTTTATATCTTGA

 1712

 ATGGTTTTTATGGCATCTATTTTTGTTAGATAGGGTT

AGATTG CTAG GC ATCTTTATCG GTATTTTG AC GCAATTCAT

 1713

 CAAAACTTCCCCAACGTGTTTTGTCTGCTGGAATTTCT

ACACCTTCTTGTGCATTAACAGCCAAGCTTGCTAGCGAAAA

 1714

AACTGCTAATGAACCAGCAGTTATAATTTTTTTTTTCAT The sequences of adapters A (SEQ ID No.1), and B (SEQ ID No.2) were added to each end of the probes for PCR amplification, leading to probes whose sequence is of the type " ATCGCACCAGCGTGT-N80-CACTGCGGCTCCTCA ", where N80 corresponds to the specific sequence of each of the 4 probes. Probes with a total length of 1 10 nucleotides were synthesized as single-stranded DNA. The T7 promoter (SEQ ID No.3) was added upstream of adapter A by PCR with the Platinium Taq DNA High Fidelity Polymerase Kit (Invitrogen) using primers T7-A and B (SEQ ID No. 1715 and 1716) respectively hybridizing to Adapters A and B (SEQ ID Nos. 1 and 2). The PCR products obtained were purified with the MinElute PCR purfication kit (Qiagen). Biotinylated single-stranded RNA probes were synthesized by in vitro transcription using the MEGAScript kit (Ambion) and biotinylated dUTPs (Tebu- Bio), and were purified with the RNeasy plus kit (Qiagen).

Cl Preparation of large DNA fragment libraries: For the construction of the library, 4 μg DNA of the DNA mixture of the artificial community was fragmented to a size of 20 kb using the kit g-TUBE (Covaris ). Fragments of a size of 20 kb (+/- 4kb) were selected with the BluePippin system (Sage Science) and then amplified using the Illustra GenomPhi V2 DNA Amplification Kit (ref 25-6600-32, GE Healthcare). Amplified fragments of 20 kb (+/- 4kb) size were again selected with the BluePippin (Sage Science) system to obtain a DNA library of large fragments of homogeneous size. The quality of the library was finally assessed by Qubit DNA assay (Life Technologies) and Agilent DNA 12000 Agile Migration (Agilent Technologies).

PI Hybridization and Capture: To carry out the hybridization, 3 μg of the library were mixed with 2.5 μg of salmon sperm DNA (Salmon Sperm DNA, sheared (ref AM9680, Ambion)) and denatured for 5 minutes at 95 ° C. ° C and then incubated for 5 minutes at 65 ° C. At the end of the incubation, 13 μl of hybridization buffer (10 mol / L SSPE, Denhardt's 10 mol / L, 10 mM EDTA, pH 8 and 0.2% SDS) and then 500 ng of biotinylated RNA probes preheated to 65 ° C. C were added to the mixture. After 24 hours of hybridization at 65 ° C, probe / library hybridization complexes were captured using 500 ng of streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin (ref 1205D, Life Technologies)) previously washed three times. once with 200 L of 1 M NaCl / 10 mM TE. The beads were washed three times at room temperature with 500 μl of 1 × SSC / 0.1% SDS, then three times at 65 ° C. with 500 μl of 0.1 × SSC / 0.1% preheated SDS. The captured DNA fragments were then eluted with 50 μl of 0.1 M NaOH. After the sedimentation of the beads, the supernatant containing the enriched DNAs was transferred to a tube containing 70 μl of 1 M Tris-HCl, pH 7. 5. The captured DNA fragments were then purified using the Microcon DNA Fast Flow PCR Grade Kit, Centrifugal Filters, Dual Cycle ΕΤ0 Treated (ref MRCF0R100ET, Merck Millipore) and amplified using the Illustra GenomPhi V2 DNA Amplification Kit (ref. 6600-32, GE Healthcare). Finally, the amplified fragments of a size of 20 kb (+/- 4 kb) were selected with the BluePippin system (Sage Science). Sequencing of the captured DNA fragments

The captured DNA fragments were sequenced on 1/4 of Illumina's MiSeq 2x300 bp sequencing run, after a prior step of constructing the Nextera (Illumina) sequencing library in accordance with the manufacturer's instructions. .

FI Processing of the Sequencing Data The readings obtained following the sequencing of the enriched DNAs were filtered according to their quality using the PRINSEQ-lite script (Schmeider, Bioinformatics, 201 1). Thus 5 166 235 pairs of sequences were obtained. Readings were de novo with IDBA-UD v1.1.2 (Peng, Bioinformatics, 2012). The contigs obtained were then subjected to a second assembly using the CAP3 tool (Huang, Genome Research, 1999). Contigs carrying the target gene were identified by BLASTN (AltschuI, Journal of Molecular Biology, 1990) and then affiliated using BLASTN against the reference genome base (06/10/14) of NCBI. Finally, the readings were aligned with Bowtie2 v2.1.0 (Langmead, Nature Methods, 2012) against the reference genome of C. glutamicum.

Gl Results obtained

The present method allowed the complete reconstruction of the targeted 2061 bp gene encoding the F. psychrophilum pepO gene but also its flanking regions. Indeed, a contig 7.1 kb corresponding to a portion of the genome of F. psychrophilum carrying the gene of interest could be reconstructed. This was made possible by the targeted enrichment of the gene and adjacent regions which account for 10% of the sequences whereas the genome of F. psychrophilum initially represented only 0.01% of the community and the reconstructed DNA region represented than 0.00005% of all sequences in the microbial community. The enrichment of the target sequence allowing the characterization of the micro-organism is therefore of the order of 200 000 times. Thus, this pathogenic microorganism causing massive mortality in these salmonid species could be highlighted in the community studied. When applied to contaminated fish tissue, this method can be used to detect the pathogen in a controlled manner even if the pathogen is present in very small quantities. Applied to freshwater samples, this strategy can detect and identify precisely F. psychrophilum among the bacterial communities present to implement appropriate prevention strategies, and thus reduce the occurrence of flavobacteriosis resulting in losses. important economic factors in fish farming.

Claims

A method for the in vitro detection and identification of one or more target pathogens present in a biological sample, said method comprising the steps of:  a) preparing a mixture of probes capable of specifically targeting a set of gene sequences of interest belonging to the target pathogens,  b) preparing a library of DNA fragments from the biological sample, said DNA fragments contained in the library having a length of at least 6,000 base pairs,  c) hybridizing the DNA fragments obtained in step b) by contacting said DNA fragments with the mixture of probes of step a), d) capturing the hybridization complexes of the step c),  e) sequencing the DNA fragments captured in step d), and  f) bioinformatic reconstruction of the genes of interest and / or the genome of the microorganism or microorganisms from the captured DNA fragments sequenced from step e), and  (g) identification of the target pathogen (s) present in the biological sample. The detection and identification method according to claim 1, wherein the target pathogen (s) are selected from the group consisting of: bacteria, viruses, viroids, fungi, unicellular eukaryotes and worms. The detection and identification method according to claim 2, wherein the target bacteria are selected from the group consisting of: Bacillus anthracis, Clostridium botulinum, Francisella tularensis, Yersinia pestis, Brucella, Burkholderia, Campylobacter jejuni, Chlamydiophila psittaci, Coxiella burnetii , Escherichia coli, Clostridium perfringens, Listeria monocytogenes, Vibrio, Salmonella, Shigella, Staphylococcus aureus, Rickettsia, Yersinia enterocolitica, Mycobacterium tuberculosis, Anaplasma phagocytophilum, Bartonella henselae, Bordetella pertussis, Borrelia miyamotoi, Clostridium difficile, Ehrlichia, Enterococcus, Leptospira, Borrelia burgdorferi, Streptococcus pyogenes, Legionella, Mycoplasma, Nocardia, Pneumocystis or a mixture of two or more of these target bacteria. 4. A method of detection and identification according to any one of claims 1 to 3, wherein the biological sample can be obtained from a body fluid or a tissue taken from the animal or from the Man or in plants. 5. detection and identification method according to any one of claims 1 to 4, wherein the gene sequences of interest are sequences corresponding to the virulence genes, antibiotic resistance genes, phylogenetic markers, functional markers or repeated sequences. The detection and identification method according to any one of claims 1 to 5, wherein the probes of step a) are single-stranded RNA probes. The detection and identification method according to any of claims 1 to 6, wherein the probes of step a) are at least 20 contiguous nucleotides in length. The method of detection and identification according to any one of claims 1 to 7, wherein the probes of step a) are non-overlapping and exploratory probes. The detection and identification method according to any one of claims 1 to 8, wherein the probes of step a) are biotinylated RNA probes. 10. Detection and identification method according to any one of claims 1 to 9, wherein the probes of step a) comprises an adapter A sequence SEQ ID No.1 and an adapter B sequence SEQ ID No .2, the adapter A being placed at the 5 'end of the probe sequence and the adapter B being placed at the 3' end of the probe sequence. 1 1. A detection and identification method according to any one of claims 1 to 10, wherein the probe mixture of step a) comprises at least one probe. 12. The method of detection and identification according to any one of claims 1 to 1 1, wherein the probe mixture is specific for at least 1 gene of interest. The detection and identification method according to any one of claims 1 to 12, wherein the library of DNA fragments comprises DNA fragments having a length of at least 20,000 base pairs, preferably at least less than 50,000 base pairs. 14. The method of detection and identification according to any one of claims 1 to 13, wherein the c) hybridization step is carried out in solution. The method of detection and identification according to any one of claims 1 to 14, wherein the step d) of capturing the hybridization complexes is carried out using magnetic beads coated with streptavidin.