WO2012101377A1 - Multifunctional diagnostic device - Google Patents

Multifunctional diagnostic device Download PDF

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Publication number
WO2012101377A1
WO2012101377A1 PCT/FR2012/050156 FR2012050156W WO2012101377A1 WO 2012101377 A1 WO2012101377 A1 WO 2012101377A1 FR 2012050156 W FR2012050156 W FR 2012050156W WO 2012101377 A1 WO2012101377 A1 WO 2012101377A1
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subject
gene
dna
stool
diagnostic device
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PCT/FR2012/050156
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French (fr)
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Jean-Pierre Roperch
Iradj Sobhani
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Assistance Publique Hopitaux De Paris
Profilome
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Publication of WO2012101377A1 publication Critical patent/WO2012101377A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon

Definitions

  • the present invention relates to a multifunctional diagnostic device, particularly relevant for the diagnosis of digestive diseases as well as for screening for colorectal cancer in a subject.
  • Occult bleeding tests are currently the most commonly used procedure for screening for colorectal cancer in large, asymptomatic populations. Among them, tests based on the use of monoclonal or polyclonal antibodies recognizing the globin part of hemoglobin appear to be more efficient than traditional tests using guaiac resin. Nevertheless, this type of screening test suffers from a number of disadvantages. The major disadvantage of this type of test is its low sensitivity, leaving an unacceptable number of false negatives among the subjects subjected to said test which while carrying a tumor will not be detected due to the absence of bleeding. continuous colorectal tumors. The specificity of this test also remains mediocre.
  • the appearance of blood in the stool may be related to a non-tumoral condition: gastric, ileal, or recto-colic haemorrhage of non-tumoral origin, hemorrhoidal or superficial anal lesions.
  • gastric, ileal, or recto-colic haemorrhage of non-tumoral origin hemorrhoidal or superficial anal lesions.
  • nearly 50% of colonoscopies performed in individuals with a positive occult blood test are normal.
  • nearly 50% of subjects with colonic, benign or malignant tumors are not identified as such by this screening strategy.
  • the inventors have succeeded in implementing a molecular test that makes it possible to identify several markers of interest whose hypermethylation is specific to colorectal cancers.
  • the present invention is specifically intended to meet this need. Indeed, surprisingly and unexpectedly, the inventors have succeeded in adapting said media currently used in immunological diagnostic kits to develop a single diagnostic device, allowing the simultaneous or sequential combination of two methods for diagnosing the diagnosis. colorectal cancer in a subject.
  • the inventors have developed a diagnostic device particularly adapted to the implementation of several diagnostic tests, based on different approaches. It allows the simultaneous implementation of several diagnostic tests on a single biological sample obtained from a subject.
  • this biological sample is constituted by the stool of said subject in search of a diagnosis.
  • the invention relates to a diagnostic device comprising:
  • a lysis solution optionally, a lysis solution
  • Such a device allows, at first, the implementation of an immunological test by the detection and measurement of occult blood in the stool of a subject. Such a presence is correlated with the presence or the risk of occurrence of cancerous conditions in said subject.
  • the device of the invention allows, in a second time, the implementation of a molecular test.
  • This test can take several forms:
  • the device according to the invention makes it possible to detect the presence of a mutation of a marker gene for colorectal cancer, preferably a gene chosen from the Kras gene and the APC gene, or to determine a genetic instability at the level of DNA microsatellite repeat sequences selected from Bat25 and Bat26, and / or
  • markers of colorectal cancer are in particular the WifI, NPY, PENK genes; and or
  • the detection and measurement of occult blood in the stool is performed with the device according to the invention by means of an immunological test.
  • This test makes it possible to detect the presence and to measure the concentration of occult blood in the stool of a subject using antibodies specific for hemoglobin, preferentially hemoglobin globin.
  • Such antibodies may be polyclonal or monoclonal antibodies. The general knowledge of the skilled person allows him to obtain such antibodies specific for hemoglobin.
  • Subject means a healthy individual or an individual who may be suffering from cancer or in search of screening, diagnosis or follow-up, and more generally any individual with a subjective or physical symptom leading health care workers to use diagnostic tests.
  • subject also refers to healthy animals or for which a diagnosis or screening is necessary.
  • diagnosis is meant the detection of a predisposition to develop a pathology, but also the detection of a pathology already installed in a subject.
  • immunological diagnosis or “immunological test” is meant a diagnostic test based on the search for occult blood in the stool of a subject using antibodies specific for hemoglobin.
  • the polyclonal antibodies can be obtained by immunizing an animal against the antigen of interest, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by affinity chromatography on a column on which is fixed an antigen specifically recognized by the antibodies.
  • the term "antigen of interest” is understood to mean hemoglobin or, preferably, globin. This hemoglobin or globin may be human or animal depending on the nature of the subject to be tested.
  • Monoclonal antibodies can be obtained by the hybridoma technique (Kohler and Milstein, 1975).
  • the monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.
  • globin specific antibodies to hemoglobin are known and are widely available commercially. Such antibodies are for example marketed by the companies EIKEN and INTEX Diagnostika. The society EIKEN also markets a device that allows the detection of occult blood in the stool of a subject using antibodies specific for hemoglobin.
  • the antibody specific for hemoglobin is labeled.
  • labeling is meant the attachment of a reagent capable of directly or indirectly generating a detectable signal.
  • a non-limiting list of these reagents consists of:
  • enzymes that produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent compounds, luminescent compounds, dyes, radioactive molecules such as 3 J 2 "P, 3 J 5 J S ⁇ 25 or 1 J I, and
  • fluorescent molecules such as Alexa or phycocyanins.
  • the antibodies specific for hemoglobin, and more preferably globin can be immobilized on a solid support adapted within the diagnostic device according to the invention or placed in suspension in a suitable medium.
  • the device according to the invention comprises a solid support on which the antibodies specific for hemoglobin are fixed.
  • the antibodies are immobilized on a solid support within the diagnostic device according to the invention.
  • Said solid support may be made of glass, or plastic, in particular polystyrene or nitrocellulose.
  • the antibodies are adsorbed spontaneously and irreversibly on the membrane. They can therefore be attached to the wall of the device, or to moving particles within said device. Any material not bound to the antibody can be removed by widely used techniques such as centrifugation, decantation of the supernatant, or elution.
  • the device according to the invention comprises an amount of antibody sufficiently high to allow effective detection of the hemoglobin possibly present in the stools of the test subjects.
  • the skilled person is able to adapt this amount of antibody depending on the amount of stool used for the implementation of the immunoassay.
  • the device according to the invention for the implementation of a molecular test
  • the diagnostic device allows, in a second step, the implementation of a so-called molecular test.
  • This test allows a total DNA extraction to allow the detection and the quantification of possible modifications of the DNA of the cells of said subject and the characterization of the bacterial DNA of said subject.
  • Immunological and molecular tests can be performed simultaneously or sequentially.
  • the genomic DNA is extracted between 24 h and 72 h, preferably 48 h after the stool of the test subject is brought into contact with the diagnostic device according to the invention.
  • molecular diagnostics or “molecular test” is meant a test based on the analysis of the genomic DNA of the subject or of the bacterial DNA originating from faecal bacteria present in the stool of said subject to be tested.
  • this term applies to diagnostic tests based on the direct analysis of the sequences of all or part of the DNA of interest and / or after amplification of a part of DNA of interest and analysis of methylation levels of marker genes for colorectal cancer, identified by the inventors.
  • this expression applies to any diagnostic test based on the analysis of human or bacterial DNA markers specific for digestive diseases.
  • This expression thus encompasses the identification and quantification of a bacterial population in colon cancer (Sobhani et al., PloS One, 2011 in press), Crohn's disease (Sokol et al, Gut, 2007 Jan; 56 (1)). ): 152-4, Seksik et al, J Clin Microbiol 2005 Sep; 43 (9): 4654-8; Sokol H, et al; Proc Natl Acad Sci US A, 2008 Oct 28; 105 (43): 16731 -6), in diseases of the liver and bile ducts (Abu-Shanab A, Quigley EM.Nat Rev Gastroenterol Hepatol, 2010 Dec; 7 (12): 691-701) or in extra digestive diseases such as obesity or diabetes (Furet etal; Diabetes, 2010 Dec; 59 (12): 3049-57).
  • total DNA is meant without distinction genomic and bacterial DNA.
  • genomic DNA is meant DNA from cells of a subject, usually epithelial cells of that subject. This expression applies in particular to the DNA of exfoliated intestinal cells found in the stool of the test subject. This DNA is extracted and purified and amplified by PCR. The amplification products are then analyzed for specific mutations of cancer pathologies.
  • bacterial DNA or "plasmid DNA” is meant DNA from the bacteria present in the biological sample obtained from said subject. This expression applies in particular to the DNA of the bacteria originating from the intestinal flora of the subject to be tested. It is known that 80% of faecal bacteria remain uncultivable. Also, the device of the invention has the advantage of facilitating their detection.
  • DNA extraction is meant any technique for isolating genomic or bacterial DNA from a biological sample, preferably from stool of a subject to be tested. In the context of this invention is meant a technique for isolating the DNA obtained from the stool of the subject.
  • the DNA thus extracted may be genomic DNA or bacterial DNA.
  • the DNA can then be used for molecular diagnostic purposes, in particular for the diagnosis of cancerous pathologies, such as colorectal cancers. There are different protocols for extracting DNA, which follow approximately the same basic scheme:
  • lysis of the cells generally using a lysis solution, conventionally used in the prior art and widely described, such as in the context of the so-called phenol-chloroform extraction method;
  • Those skilled in the art may refer to commercial kits, such as the QIAamp DNA Stool kit (Qiagen) and allowing easy, efficient and reproducible extraction of genomic DNA.
  • kits such as the QIAamp DNA Stool kit (Qiagen) and allowing easy, efficient and reproducible extraction of genomic DNA.
  • the DNA lysis and extraction solutions allow the implementation of a DNA extraction of a biological sample, preferably the stool of the test subject, according to the techniques conventionally described in the prior art.
  • This extraction can be done by a first cell lysis step. Lysis can be done in different ways: mechanical dislocation, hypotonic shock, chemical lysis, or digestion with proteinase K. Preferably, this lysis is a chemical lysis step.
  • the lysis solution present in the diagnostic device according to the invention is a mixture of detergent and proteinase K.
  • a mixture makes it possible to dissociate the cells, the tissues, the envelopes of the viruses and to release the nucleic acids.
  • the currently most used detergents are sodium dodecyl sulfate (SDS), sarcosyl, Tween® 20, nonidet.
  • the presence of the lysis solution within the device according to the invention is optional and conditions the process using said device. Also, if the device does not include lysis solution, it will be appropriate to undergo a cell lysis step to the biological sample tested, before bringing it into contact with said device.
  • genomic DNA extraction should be carried out as such.
  • this extraction step is carried out using a chaotropic agent, preferably guanidium thiocyanate (GTC).
  • GTC guanidium thiocyanate
  • the function of CTG is to disrupt cell tissue membranes and denature proteins.
  • the deletion of protein disulfide bridges, avoiding protein renaturation can be done by the addition of beta mercaptoethanol in the medium.
  • the DNA extraction solution present in the diagnostic device according to the invention is a mixture of guanidium thiocyanate and beta-2-mercaptoethanol.
  • the present invention also provides the possibility of carrying out this extraction step according to the so-called phenol-chloroform technique.
  • Phenol is indeed a powerful deproteinizing. Its addition in the medium containing the cell lysate has the effect of denaturing the proteins. After centrifugation, they are at the interface between the aqueous phase (which contains the nucleic acids) and the organic phase. After transfer of the aqueous phase to another tube, the nucleic acids are treated with a chloroform-isoamyl alcohol mixture. This step eliminates traces of phenol. This step is essential since phenol is known to be toxic but is also an inhibitor of Taq polymerase. After mixing, and centrifugation and removal of the organic phase, the nucleic acids are in the soluble state in the aqueous phase.
  • the DNA extraction solution preferably comprises guanidium thiocyanate and beta-2-mercaptoethanol.
  • guanidium thiocyanate and beta-2-mercaptoethanol are able to adapt the composition and the concentrations of guanidium thiocyanate and beta-2-mercaptoethanol.
  • Those skilled in the art can refer to the manual "Principles of molecular biology in clinical biology" by Amezian, Bogard and Lamoril, Campus Reference, Elsevier.
  • the DNA extraction solution comprises 4M guanidium thiocyanate and 0.5% beta-2-mercaptoethanol.
  • the diagnostic device according to the invention comprises beads, preferably glass beads. Typically, it is glass beads calibrated at 0.5 mm. These beads make it possible to mechanically improve the performance of the chemical action of the lysis solution. They therefore participate in the lysis of the cells present in the stool of the test subject.
  • the total volume of the beads does not exceed 1/3 of the total volume of the device according to the invention.
  • the diagnostic device according to the invention is highly advantageous because it makes it possible to combine three approaches to diagnosing colon cancer:
  • the invention further relates to a method of screening for colorectal cancer comprising:
  • step A) of the method according to the invention is carried out by means of the immunological test carried out by means of the hemoglobin-specific antibodies present in the device of the invention.
  • the stool of the subject to be tested can be brought into contact with the device directly after the sampling or after a first cell lysis step.
  • This immunological test reveals the presence of blood with great precision.
  • the biological sample is contacted with said hemoglobin-specific antibodies. Visualization of immunological reactions can be performed by any means of detection, such as direct or indirect means.
  • the direct detection can be done without the intermediary of a marking, the immunological reactions are observed for example by surface plasmon resonance or by cyclic voltammetry on an electrode carrying a conductive polymer.
  • the direct detection is via a labeling of said antibody.
  • Indirect detection systems can also be used, for example ligands capable of reacting with an anti-ligand.
  • the ligand / anti-ligand pairs are well known to those skilled in the art, such as the biotin / streptavidin pair. In this case, it is the ligand that carries the binding partner.
  • the anti-ligand can be detectable directly by the labeling reagents described in the preceding paragraph or be itself detectable by a ligand / anti-ligand.
  • hemoglobin preferably globin
  • purification is carried out, generally carried out by chromatography, for example exclusion, by affinity or by dialysis.
  • the hemoglobin concentration of the stools analyzed can then be evaluated according to the conventional techniques of the prior art.
  • the presence of occult blood in stool is correlated with a risk of presence of a cancerous pathology, such as cancer of the intestine, colon or rectum.
  • step B) of the method is carried out according to the modalities previously developed.
  • the method for detecting colorectal cancer in a subject according to the invention further comprises:
  • the method of the invention provides a step C) of determining the presence of a mutation of a marker gene for colorectal cancer.
  • this step C) is a step of determining the presence of a mutation of a gene selected from the Kras gene and the APC gene.
  • mutation is meant any phenomenon of modification of the genetic material. This term includes insertions, substitutions and deletions.
  • Kras gene is meant the gene encoding the Kras protein. Recent studies have shown that the presence of a mutation in the Kras gene is associated with a lack of clinical benefit to anti-EGFR treatments. Thus, by determining the presence or absence of mutation of the Kras gene in a subject, health actors can adapt the appropriate therapy for the subject. Indeed, only patients with a tumor with a non-mutated Kras gene can benefit from anti-EGFR treatment.
  • APC gene is meant the gene coding for the protein "Adenomatous polyposis coli”. This gene has been described as possessing tumor suppressor activity. Also, it is currently recognized that a mutation of the APC gene in a given subject, including a mutation leading to the inactivation of this gene, is associated with the existence of colorectal cancer in said subject.
  • the method of the invention provides a step D) for determining a genetic instability at the level of the DNA microsatellite repeat sequences chosen from Bat25 and Bat26.
  • the frequency of genetic alterations of the microsatellite repeat sequences of DNA can be readily determined by those skilled in the art.
  • Bat25 or “Bat26”, we designate two quasi-homozygous, quasi-monomorphic and mononucleotide microsatellites.
  • Bat25 corresponds to a repetition of thymines (poly (T) tail) and Bat26 corresponds to a repeat of adenines (poly (A) tail).
  • microsatellites short DNA sequences formed from the repetition of a motif consisting of one to four bases.
  • genetic instability at the level of the repeated DNA microsatellite sequences is characterized by the abnormal variation in the number of repeated sequences in the tumor DNA compared to the DNA of the same subject from healthy tissue.
  • the method of the invention provides a step E) for detecting hypermethylation of marker genes previously identified by the inventors and in general of any other human or bacterial DNA marker specific for digestive diseases.
  • the colorectal cancer screening method according to the invention further comprises measuring the degree of methylation of at least one of the genes selected from Wifl, NPY, PENK and their fragments or variants in the stool of said subject with the aid of the device according to the claims.
  • DNA methylation analysis can be performed using qualitative and / or quantitative PCR-based techniques such as MSP (PCR specific methylation), bisulfite sequencing, methylation-sensitive restriction enzyme digestion coupled with PCR.
  • methylation is meant the addition of a methyl group at the 5 'carbon of a cytosine in a CpG dinucleotide. These dinucleotides are infrequent in the structure of DNA, except in the CpGs "islets". These islets are typically represented at the level of the promoter region of the genes. Thus, when one speaks of methylation of a gene, reference is made to the methylation of the promoter region of said gene. The presence of a methyl group at a specific site prevents interaction between the gene and transcription factors. Typically, methyl groups prevent transcription factors from binding to the amplification site and promoter, and RNA polymerase from binding to the initiation site.
  • methylation of the promoter region results in the repression of the transcription of the DNA.
  • methylation of a gene encompasses the methylation of the CpG islets of the nucleotide sequence of the gene but also the methylation of the nucleotide sequences of the promoter of the gene to which this expression applies.
  • Multiplex PCR is meant a form of PCR, generally a quantitative PCR allowing the simultaneous amplification of several targets of interest in a single step, using one or more specific primers. This technique is very advantageous for determining the presence of deletions, mutations, polymorphisms or hypermethylation of several markers.
  • telomere PCR refers to a form of PCR, usually a quantitative PCR, allowing the amplification of a single target of interest.
  • PCR specific methylation is meant a conventional technique of the prior art for measuring the degree of methylation of a gene. This technique is based on the principle of quantitative PCR. Typically, this technique relies on treatment with sodium bisulfite of the DNA sample to be studied. This treatment makes it possible to transform each of the unmethylated cytosines in uracils in the treated DNA. The sample thus treated then undergoes PCR with primers specific to the genes to be treated. The determination of the nature of the specific primers depends on the nucleotide sequence to be amplified. In the context of this invention, the specific PCR methylation is preferably carried out in multiplex mode, which is then referred to as PCR-specific methylation in multiplex mode, ie MSPM.
  • Wifl gene is meant the gene that encodes the WIF protein acting as an inhibitor of the Wnt pathway.
  • the initiating element of colorectal carcinogenesis is the inactivation of the APC gene whose protein plays a role in controlling the proliferation, apoptosis and migration of intestinal epithelial cells. This inactivation causes the activation of certain signaling pathways, such as that of WNT / beta-catenin and the formation of early adenomas. It is known that Wifl is one of the genes involved in the inhibition of the Wnt signaling pathway. Also, the hypermethylation of the Wifl gene is accompanied by the inhibition of inhibition of this pathway.
  • NPY gene is meant the gene which encodes neuropeptide Y or
  • PENK gene is meant the gene which codes for the neuropeptide proenkephalin A or PENK.
  • fragment of a gene is meant a sequence of said gene having a length of at least 50 base pairs, preferably of a length of between 60 and 110 base pairs.
  • Percentage of identity between two nucleic acid sequences or in the sense of the present invention, is meant to designate a percentage of nucleotides between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their entire length.
  • Best alignment or “optimal alignment” is meant the alignment for which the percentage of identity determined as hereinafter is the highest. Sequence comparisons between two nucleic acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being performed by segment or by "comparison window” to identify and compare the local regions of sequence similarity. .
  • the optimal alignment of the sequences for comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970) ), using the Pearson-Lipman (1988) similarity search method, using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • the BLAST program is preferably used with the BLOSUM 62 matrix. The percentage identity between two nucleic acid sequences is determined by comparing these two optimally aligned sequences.
  • nucleic acid sequence to be compared which may comprise additions or deletions with respect to the reference sequence for optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical positions for which the nucleotide is identical between the two sequences, dividing this number of identical positions by the total number of positions compared and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
  • variants having an identity percentage of at least 80%, preferably at least 90%, more preferably at least 98% are meant nucleic sequences having, relative to the sequence reference nuclei, certain modifications such as in particular deletion, truncation, elongation, chimeric fusion, and / or substitution, in particular punctual, and whose nucleic sequence has at least 80, preferably at least 90, more preferred at least 98, identity after optimal alignment with the reference nucleic sequence.
  • This step E) of the method according to the invention may also comprise the measurement of the degree of methylation of a household gene.
  • this step of measuring the degree of methylation of the household gene is carried out simultaneously to measure the degree of methylation of at least one of the genes selected from Wifl, NPY, PENK and their fragments or variants and / or simultaneously with measuring the degree of methylation of the Wif1 gene or a fragment or variant thereof.
  • housekeeping gene or “housekeeping gene” or “constitutive gene” or “housekeeping gene” is meant a constitutional gene that encodes mainly for proteins essential to basic cellular functions.
  • the expression of the housekeeping genes according to the invention is ubiquitous and stable between the different tissues.
  • the household gene is chosen from the genes encoding albumin, beta-actin, alpha-actin, beta-microglobulin, and preferably albumin. These household genes are of major interest in the context of the invention. Indeed, the degree of methylation of the NPY, PENK and Wif 1 genes or their fragments or variants is expressed with respect to the methylation of a household gene whose expression is stable.
  • degree of methylation or “level of methylation” is meant the percentage of the number of methylated CpG sites of the target gene compared to a standard.
  • target gene is meant a gene for which it is desired to measure the degree of methylation.
  • these target genes are WifI, NPY, PENK, albumin and their fragments or variants.
  • standard is meant the wild-type sequence of said target gene or the sequence of a household gene.
  • the degree of methylation is calculated according to the quantification techniques well known to those skilled in the art. This quantification can be absolute or relative. Preferably, its calculation is done according to the so-called AACt technique well known in the prior art.
  • the method of the invention also comprises a step of comparing the methylation levels on the one hand of the Wifl genes and of a household gene, or their fragments or variants and, on the other hand, of the NPY genes and PENK or their fragments or variants at a threshold value, said threshold value being determined according to the needs in terms of specificity and sensitivity.
  • step F) for identifying and quantifying a faecal bacterial profile characteristic of the existence of cancer.
  • Colorectal is a step of identifying and quantifying a bacterial profile of bacteria belonging to the genera Bacteroides or Prevotella.
  • the inventors have demonstrated a change in the colonic flora of subjects suffering from colorectal cancers, including an increase in the amount of bacteria belonging to the genera Bacteroides or Prevotella.
  • this bacterial DNA under standard conditions well known to those skilled in the art, therefore allows the determination of the composition of the intestinal flora of the subject to be tested. More specifically, this analysis allows the skilled person to determine the amount of target bacteria relevant for the diagnosis of colorectal cancer in the stool of the subject to be tested.
  • This quantity can then be compared to a threshold value, obtained in a healthy subject, ie not suffering from digestive pathologies, in particular colorectal cancers.
  • Freshly made stools are collected in a pot or dedicated container. The subject should put his stool on a solid support and protected from any contamination by water or urine.
  • the subject must soak the piston of the device in the stool thus collected and put the piston back in its case.
  • the approximate amount of stool removed is estimated to be 10 to 20 mg of stool diluted in 2 mL of lysis and extraction solution.
  • the presence of human globin is detected and quantified in situ in dedicated reading tanks.
  • the Ag-AC immunological reaction is carried out by contacting 50 ⁇ l of the antibody solution with a volume of 50 ⁇ l of biological solution. Both solutions are removed and introduced into the reaction tank in a manual or automatic mode. The reading is done automatically and directly within said reading tank according to the turbidimetric opacity process: the hemoglobin complexed with the antibody forms an agglomerate proportional to the concentration of globin. The optical reading will specify the corresponding point on a standard curve.

Abstract

The invention relates to a diagnostic device including: antibodies specific to hemoglobin, preferably to globin; optionally, a lysis solution; a solution for extracting genomic and/or bacterial DNA; and glass beads.

Description

Dispositif de diagnostic multifonctionnel  Multifunctional diagnostic device
La présente invention concerne un dispositif de diagnostic multifonctionnel, particulièrement pertinent pour le diagnostic des maladies digestives ainsi que pour le dépistage du cancer colorectal chez un sujet. The present invention relates to a multifunctional diagnostic device, particularly relevant for the diagnosis of digestive diseases as well as for screening for colorectal cancer in a subject.
Les tests de recherche de saignement occulte dans les selles constituent actuellement la procédure la plus utilisée pour un dépistage du cancer colorectal organisé dans des grandes populations asymptomatiques. Parmi eux, les tests basés sur l'utilisation d'anticorps monoclonaux ou polyclonaux reconnaissant la partie globine de l'hémoglobine apparaissent plus performants que les tests traditionnels utilisant la résine de gaïac. Néanmoins, ce type de test de dépistage souffre d'un certain nombre d'inconvénients. L'inconvénient majeur de ce type de test est sa faible sensibilité, laissant un nombre inacceptable de faux négatifs parmi les sujets soumis audit test qui tout en étant porteurs d'une tumeur ne seront pas détectés en raison de l'absence d'un saignement continu des tumeurs colorectales. La spécificité de ce test reste également médiocre. En effet, l'apparition de sang dans les selles peut être liée à une affection non tumorale: hémorragies gastrique, iléale, ou recto-coliques d'origine non tumorale, lésions anales hémorroïdaires ou fis suraires. Aussi, dans les conditions actuelles du dépistage, près de 50% des coloscopies réalisées chez les individus ayant un test de recherche de sang occulte positive, sont normales. D'autre part, près de 50% des sujets porteurs d'une tumeur colique, bénigne ou maligne, ne sont pas identifiés comme tels par cette stratégie de dépistage. De façon surprenante, les inventeurs ont réussi à mettre en œuvre un test moléculaire permettant d'identifier plusieurs marqueurs d'intérêt dont l'hyperméthylation est spécifique des cancers colorectaux. Occult bleeding tests are currently the most commonly used procedure for screening for colorectal cancer in large, asymptomatic populations. Among them, tests based on the use of monoclonal or polyclonal antibodies recognizing the globin part of hemoglobin appear to be more efficient than traditional tests using guaiac resin. Nevertheless, this type of screening test suffers from a number of disadvantages. The major disadvantage of this type of test is its low sensitivity, leaving an unacceptable number of false negatives among the subjects subjected to said test which while carrying a tumor will not be detected due to the absence of bleeding. continuous colorectal tumors. The specificity of this test also remains mediocre. Indeed, the appearance of blood in the stool may be related to a non-tumoral condition: gastric, ileal, or recto-colic haemorrhage of non-tumoral origin, hemorrhoidal or superficial anal lesions. Also, under current screening conditions, nearly 50% of colonoscopies performed in individuals with a positive occult blood test are normal. On the other hand, nearly 50% of subjects with colonic, benign or malignant tumors are not identified as such by this screening strategy. Surprisingly, the inventors have succeeded in implementing a molecular test that makes it possible to identify several markers of interest whose hypermethylation is specific to colorectal cancers.
Ce test moléculaire, combiné au test immunologique de détection de sang occulte offre des perspectives avantageuses au dépistage des cancers colorectaux. Cependant, la mise en œuvre de ces deux tests simultanément ou séquentiellement peut s'avérer longue, fastidieuse et coûteuse. En outre, les kits de diagnostic actuellement sur le marché ne sont pas adaptés à la mise en œuvre simultanée ou séquentielle d'un test immunologique et du test moléculaire développé par les inventeurs. En effet, le matériel génétique présent dans les tissus ou les cellules à analyser sont sujets à des contaminations, notamment bactériennes, si ces dernières sont maintenues dans les milieux classiquement utilisés dans les kits de diagnostic immunologique permettant la détection de sang occulte.  This molecular test, combined with the immunological test for the detection of occult blood, offers advantageous prospects for screening for colorectal cancers. However, the implementation of these two tests simultaneously or sequentially can be long, tedious and expensive. In addition, diagnostic kits currently on the market are not suitable for the simultaneous or sequential implementation of an immunological test and the molecular test developed by the inventors. Indeed, the genetic material present in the tissues or cells to be analyzed are subject to contaminations, especially bacterial, if they are maintained in the media conventionally used in immunological diagnostic kits for the detection of occult blood.
Il existe donc un besoin pour un test simple et efficace permettant d'écarter les faux négatifs et les faux positifs, se basant sur la mise en œuvre de plusieurs techniques de dépistage de différente nature, à partir d'un seul et même échantillon du sujet à tester.  There is therefore a need for a simple and effective test to rule out false negatives and false positives, based on the implementation of several screening techniques of different kinds, from a single sample of the subject. to test.
La présente invention a précisément pour but de répondre à ce besoin. En effet, de manière surprenante et inattendue, les inventeurs ont réussi à adapter lesdits milieux actuellement utilisés dans les kits de diagnostic immunologique pour mettre au point un seul et unique dispositif de diagnostic, permettant la combinaison simultanée ou séquentielle de deux méthodes pour le diagnostic du cancer colorectal chez un sujet. Les inventeurs ont développé un dispositif de diagnostic particulièrement adapté à la mise en œuvre de plusieurs tests de diagnostic, se basant sur des approches différentes. Il permet la mise en œuvre simultanée de plusieurs tests de diagnostic sur un seul et même échantillon biologique obtenu à partir d'un sujet. Préférentiellement, cet échantillon biologique est constitué par les selles dudit sujet en quête d'un diagnostic. The present invention is specifically intended to meet this need. Indeed, surprisingly and unexpectedly, the inventors have succeeded in adapting said media currently used in immunological diagnostic kits to develop a single diagnostic device, allowing the simultaneous or sequential combination of two methods for diagnosing the diagnosis. colorectal cancer in a subject. The inventors have developed a diagnostic device particularly adapted to the implementation of several diagnostic tests, based on different approaches. It allows the simultaneous implementation of several diagnostic tests on a single biological sample obtained from a subject. Preferably, this biological sample is constituted by the stool of said subject in search of a diagnosis.
Ainsi, l'invention concerne un dispositif de diagnostic comprenant :  Thus, the invention relates to a diagnostic device comprising:
des anticorps spécifiques de l'hémoglobine, préférentiellement de la globine;  antibodies specific for hemoglobin, preferentially globin;
optionnellement, une solution de lyse;  optionally, a lysis solution;
une solution d'extraction d'ADN génomique ou bactérien; et des billes de verre.  a genomic or bacterial DNA extraction solution; and glass beads.
Un tel dispositif permet, dans un premier temps, la mise en œuvre d'un test immunologique par la détection et la mesure de sang occulte dans les selles d'un sujet. Une telle présence est corrélée avec la présence ou le risque d'apparition de pathologies cancéreuses chez ledit sujet.  Such a device allows, at first, the implementation of an immunological test by the detection and measurement of occult blood in the stool of a subject. Such a presence is correlated with the presence or the risk of occurrence of cancerous conditions in said subject.
Le dispositif de l'invention permet, dans un second temps, la mise en œuvre d'un test moléculaire. Ce test peut prendre plusieurs formes:  The device of the invention allows, in a second time, the implementation of a molecular test. This test can take several forms:
i) il permet de détecter et de quantifier d'éventuelles modifications de l'ADN des cellules dudit sujet. Plus spécifiquement, le dispositif selon l'invention permet de détecter la présence d'une mutation d'un gène marqueur du cancer colorectal, préférentiellement un gène choisi parmi le gène Kras et le gène APC, ou de déterminer une instabilité génétique au niveau des séquences répétées microsatellites de l'ADN choisies parmi Bat25 et Bat26;et/ou i) it makes it possible to detect and quantify any modifications of the DNA of the cells of said subject. More specifically, the device according to the invention makes it possible to detect the presence of a mutation of a marker gene for colorectal cancer, preferably a gene chosen from the Kras gene and the APC gene, or to determine a genetic instability at the level of DNA microsatellite repeat sequences selected from Bat25 and Bat26, and / or
ii) il permet également d'évaluer le degré de méthylation de plusieurs gènes que les inventeurs ont identifié comme marqueurs de cancer colorectaux. Ces marqueurs sont notamment les gènes Wifl, NPY, PENK; et/ou  ii) it also makes it possible to evaluate the degree of methylation of several genes that the inventors have identified as markers of colorectal cancer. These markers are in particular the WifI, NPY, PENK genes; and or
iii) il permet en outre d'identifier et quantifier un profil bactérien fécal particulièrement caractéristique de l'existence d'un cancer colorectal.  iii) it also makes it possible to identify and quantify a faecal bacterial profile that is particularly characteristic of the existence of colorectal cancer.
Le dispositif selon l'invention pour la mise en œuyre d'un test immunologique The device according to the invention for the implementation of an immunological test
La détection et la mesure de sang occulte dans les selles est réalisée avec le dispositif selon l'invention par le biais d'un test dit immunologique. Ce test permet de détecter la présence et mesurer la concentration de sang occulte dans les selles d'un sujet à l'aide d'anticorps spécifiques de l'hémoglobine, préférentiellement de la globine de l'hémoglobine. De tels anticorps peuvent être des anticorps polyclonaux ou monoclonaux. Les connaissances générales de l'homme du métier lui permettent d'obtenir de tels anticorps spécifiques de l'hémoglobine.  The detection and measurement of occult blood in the stool is performed with the device according to the invention by means of an immunological test. This test makes it possible to detect the presence and to measure the concentration of occult blood in the stool of a subject using antibodies specific for hemoglobin, preferentially hemoglobin globin. Such antibodies may be polyclonal or monoclonal antibodies. The general knowledge of the skilled person allows him to obtain such antibodies specific for hemoglobin.
Par "sujet", on entend un individu sain ou un individu susceptible d'être atteint d'un cancer ou en quête de dépistage, de diagnostic ou de suivi, et plus généralement tout individu présentant un symptôme subjectif ou physique conduisant des acteurs de santé à recourir à des tests de diagnostic. Le terme sujet désigne également des animaux sains ou pour lesquels un diagnostic ou dépistage s'avère nécessaire. Par "diagnostic", on entend la détection d'une prédisposition à développer une pathologie, mais aussi la détection d'une pathologie déjà installée chez un sujet. "Subject" means a healthy individual or an individual who may be suffering from cancer or in search of screening, diagnosis or follow-up, and more generally any individual with a subjective or physical symptom leading health care workers to use diagnostic tests. The term subject also refers to healthy animals or for which a diagnosis or screening is necessary. By "diagnosis" is meant the detection of a predisposition to develop a pathology, but also the detection of a pathology already installed in a subject.
Par "diagnostic immunologique" ou "test immunologique", on entend un test de diagnostic basé sur la recherche de sang occulte dans les selles d'un sujet à l'aide d'anticorps spécifique de l'hémoglobine.  By "immunological diagnosis" or "immunological test" is meant a diagnostic test based on the search for occult blood in the stool of a subject using antibodies specific for hemoglobin.
Par "saignement colique", ou "sang occulte" ou "saignement occulte", on désigne la présence de sang dans les selles d'un sujet.  By "colonic bleeding" or "occult blood" or "occult bleeding" is meant the presence of blood in the stool of a subject.
Typiquement, les anticorps polyclonaux peuvent être obtenus par immunisation d'un animal contre l'antigène d'intérêt, suivie de la récupération des anticorps recherchés sous forme purifiée, par prélèvement du sérum dudit animal, et séparation desdits anticorps des autres constituants du sérum, notamment par chromatographie d'affinité sur une colonne sur laquelle est fixé un antigène spécifiquement reconnu par les anticorps. Dans le cadre de cette invention, par "antigène d'intérêt", on entend l'hémoglobine ou préférentiellement la globine. Cette hémoglobine ou globine peut être humaine ou animale en fonction de la nature du sujet à tester.  Typically, the polyclonal antibodies can be obtained by immunizing an animal against the antigen of interest, followed by the recovery of the desired antibodies in purified form, by taking the serum of said animal, and separating said antibodies from the other constituents of the serum, in particular by affinity chromatography on a column on which is fixed an antigen specifically recognized by the antibodies. In the context of this invention, the term "antigen of interest" is understood to mean hemoglobin or, preferably, globin. This hemoglobin or globin may be human or animal depending on the nature of the subject to be tested.
Les anticorps monoclonaux peuvent être obtenus par la technique des hybridomes (Kôhler et Milstein, 1975).  Monoclonal antibodies can be obtained by the hybridoma technique (Kohler and Milstein, 1975).
Les anticorps monoclonaux peuvent être également des anticorps recombinants obtenus par génie génétique, par des techniques bien connues de l'homme du métier.  The monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, by techniques well known to those skilled in the art.
Des exemples d'anticorps spécifiques de la globine de l'hémoglobine sont connus et sont largement disponibles dans le commerce. De tels anticorps sont par exemple commercialisés par les sociétés EIKEN et INTEX Diagnostika. La société EIKEN commercialise d'ailleurs un dispositif permettant la détection de sang occulte dans les selles d'un sujet à l'aide d'anticorps spécifiques de l'hémoglobine. Examples of globin specific antibodies to hemoglobin are known and are widely available commercially. Such antibodies are for example marketed by the companies EIKEN and INTEX Diagnostika. The society EIKEN also markets a device that allows the detection of occult blood in the stool of a subject using antibodies specific for hemoglobin.
Préférentiellement, l'anticorps spécifique de l'hémoglobine est marqué. Par "marquage", on entend la fixation d'un réactif capable de générer directement ou indirectement un signal détectable. Une liste non limitative de ces réactifs consiste en:  Preferably, the antibody specific for hemoglobin is labeled. By "labeling" is meant the attachment of a reagent capable of directly or indirectly generating a detectable signal. A non-limiting list of these reagents consists of:
les enzymes qui produisent un signal détectable par exemple par colorimétrie, fluorescence, luminescence, comme la peroxydase de raifort, la phosphatase alcaline, la β-galactosidase, la glucose-6-phosphate déshydrogénase, les chromophores comme les composés fluorescents, luminescents, colorants, les molécules radioactives comme le 3 J2" P, le 3 J5J S ou le 1 ^25J I, et enzymes that produce a detectable signal, for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent compounds, luminescent compounds, dyes, radioactive molecules such as 3 J 2 "P, 3 J 5 J S ^ 25 or 1 J I, and
les molécules fluorescentes telles que les Alexa ou les phycocyanines.  fluorescent molecules such as Alexa or phycocyanins.
Dans le cadre de la présente invention, les anticorps spécifiques de l'hémoglobine, et plus préférentiellement de la globine peuvent être immobilisés sur un support solide adapté au sein du dispositif de diagnostic selon l'invention ou bien placé en suspension dans un milieu adapté.  In the context of the present invention, the antibodies specific for hemoglobin, and more preferably globin can be immobilized on a solid support adapted within the diagnostic device according to the invention or placed in suspension in a suitable medium.
Dans un mode de réalisation préféré, le dispositif selon l'invention comprend un support solide sur laquelle sont fixés les anticorps spécifiques de l'hémoglobine. Les anticorps sont donc immobilisés sur un support solide au sein du dispositif de diagnostic selon l'invention. Ledit support solide peut être en verre, ou plastique, notamment en polystyrène ou en nitrocellulose. Dans ce mode de réalisation particulier, les anticorps s'adsorbent spontanément et irréversiblement sur la membrane. Ils peuvent donc être attachés à la paroi du dispositif, ou bien sur des particules mobiles au sein dudit dispositif. Tout matériel non lié à l'anticorps peut être enlevé selon des techniques largement utilisées comme la centrifugation, la décantation du surnageant, ou l'élution. In a preferred embodiment, the device according to the invention comprises a solid support on which the antibodies specific for hemoglobin are fixed. The antibodies are immobilized on a solid support within the diagnostic device according to the invention. Said solid support may be made of glass, or plastic, in particular polystyrene or nitrocellulose. In this particular embodiment, the antibodies are adsorbed spontaneously and irreversibly on the membrane. They can therefore be attached to the wall of the device, or to moving particles within said device. Any material not bound to the antibody can be removed by widely used techniques such as centrifugation, decantation of the supernatant, or elution.
De manière préférée, le dispositif selon l'invention comporte une quantité d'anticorps suffisamment élevée pour permettre une détection efficace de l'hémoglobine éventuellement présente dans les selles des sujets à tester. L'homme du métier est en mesure d'adapter cette quantité d'anticorps en fonction de la quantité de selles utilisées pour la mise en œuvre du test immunologique.  Preferably, the device according to the invention comprises an amount of antibody sufficiently high to allow effective detection of the hemoglobin possibly present in the stools of the test subjects. The skilled person is able to adapt this amount of antibody depending on the amount of stool used for the implementation of the immunoassay.
Le dispositif selon l'invention pour la mise en œuyre d'un test moléculaire The device according to the invention for the implementation of a molecular test
Le dispositif de diagnostic selon l'invention permet, dans un second temps, la mise en œuvre d'un test dit moléculaire. Ce test permet une extraction d'ADN total pour permettre la détection et la quantification d'éventuelles modifications de l'ADN des cellules dudit sujet et la caractérisation de l'ADN bactérien dudit sujet.  The diagnostic device according to the invention allows, in a second step, the implementation of a so-called molecular test. This test allows a total DNA extraction to allow the detection and the quantification of possible modifications of the DNA of the cells of said subject and the characterization of the bacterial DNA of said subject.
Les tests immunologiques et moléculaires peuvent être mis en œuvre de manière simultanée ou séquentielle. Préférentiellement, l'extraction d'ADN génomique se fait entre 24h et 72h, préférentiellement 48h après la mise en contact des selles du sujet à tester avec le dispositif de diagnostic selon l'invention.  Immunological and molecular tests can be performed simultaneously or sequentially. Preferably, the genomic DNA is extracted between 24 h and 72 h, preferably 48 h after the stool of the test subject is brought into contact with the diagnostic device according to the invention.
Par "diagnostic moléculaire" ou "test moléculaire", on entend un test basé sur l'analyse de l'ADN génomique du sujet ou de l'ADN bactérien issu des bactéries fécales présentes dans les selles dudit sujet à tester. De manière particulière, cette expression s'applique aux tests de diagnostic se basant sur l'analyse directe des séquences de tout ou une partie de l'ADN d'intérêt et/ou après amplification d'une partie d'ADN d'intérêt et l'analyse de degrés de méthylation de gènes marqueurs du cancer colorectal, identifiés par les inventeurs. D'une manière générale, cette expression s'applique à tout test de diagnostic se basant sur l'analyse de marqueurs d'ADN humain ou bactérien spécifiques des maladies digestives. Cette expression englobe donc l'identification et la quantification d'une population bactérienne dans le cancer du colon (Sobhani et al, PloS One, 2011 in press), la maladie de Crohn (Sokol et al, Gut. 2007 Jan;56(l): 152-4 ; Seksik et al, J Clin Microbiol. 2005 Sep;43(9):4654-8. ; Sokol H, et al; Proc Natl Acad Sci U S A. 2008 Oct 28; 105(43): 16731-6), dans les maladies du foie et des voies biliaires (Abu-Shanab A, Quigley EM.Nat Rev Gastroenterol Hepatol. 2010 Dec;7(12):691-701) ou encore dans les maladies extra digestives telles que l'obésité ou le diabète (Furet etal ; Diabètes. 2010 Dec;59(12):3049-57). By "molecular diagnostics" or "molecular test" is meant a test based on the analysis of the genomic DNA of the subject or of the bacterial DNA originating from faecal bacteria present in the stool of said subject to be tested. In particular, this term applies to diagnostic tests based on the direct analysis of the sequences of all or part of the DNA of interest and / or after amplification of a part of DNA of interest and analysis of methylation levels of marker genes for colorectal cancer, identified by the inventors. In general, this expression applies to any diagnostic test based on the analysis of human or bacterial DNA markers specific for digestive diseases. This expression thus encompasses the identification and quantification of a bacterial population in colon cancer (Sobhani et al., PloS One, 2011 in press), Crohn's disease (Sokol et al, Gut, 2007 Jan; 56 (1)). ): 152-4, Seksik et al, J Clin Microbiol 2005 Sep; 43 (9): 4654-8; Sokol H, et al; Proc Natl Acad Sci US A, 2008 Oct 28; 105 (43): 16731 -6), in diseases of the liver and bile ducts (Abu-Shanab A, Quigley EM.Nat Rev Gastroenterol Hepatol, 2010 Dec; 7 (12): 691-701) or in extra digestive diseases such as obesity or diabetes (Furet etal; Diabetes, 2010 Dec; 59 (12): 3049-57).
Par "ADN total", on entend sans distinction l'ADN génomique et bactérien. Par "ADN génomique", on entend l'ADN issu des cellules d'un sujet, généralement de cellules épithéliales de ce sujet. Cette expression s'applique notamment à l'ADN des cellules intestinales exfoliées qui se retrouvent dans les selles du sujet à tester. Cet ADN est extrait et purifié puis amplifié par PCR. Les produits d'amplification sont ensuite analysés à la recherche de mutations spécifiques de pathologies cancéreuses.  By "total DNA" is meant without distinction genomic and bacterial DNA. By "genomic DNA" is meant DNA from cells of a subject, usually epithelial cells of that subject. This expression applies in particular to the DNA of exfoliated intestinal cells found in the stool of the test subject. This DNA is extracted and purified and amplified by PCR. The amplification products are then analyzed for specific mutations of cancer pathologies.
Par "ADN bactérien" ou "ADN plasmidique" on entend l'ADN issu des bactéries présentes dans l'échantillon biologique obtenu à partir dudit sujet. Cette expression s'applique notamment à l'ADN des bactéries provenant de la flore intestinal du sujet à tester. Il est connu que 80% des bactéries fécales restent non cultivables. Aussi, le dispositif de l'invention présente l'avantage de faciliter leur détection. Par "extraction d'ADN", on entend toute technique permettant d'isoler l'ADN génomique ou bactérien à partir d'un échantillon biologique, préférentiellement à partir de selles d'un sujet à tester. Dans le cadre de cette invention, on entend une technique permettant d'isoler l'ADN obtenu à partir des selles du sujet. L'ADN ainsi extrait peut être de l'ADN génomique ou de l'ADN bactérien. L'ADN peut ensuite être utilisé à des fins de diagnostic moléculaire, notamment pour le diagnostic de pathologies cancéreuses, telles que les cancers colorectaux. Il existe différents protocoles pour extraire l'ADN, qui suivent approximativement le même schéma de principe : By "bacterial DNA" or "plasmid DNA" is meant DNA from the bacteria present in the biological sample obtained from said subject. This expression applies in particular to the DNA of the bacteria originating from the intestinal flora of the subject to be tested. It is known that 80% of faecal bacteria remain uncultivable. Also, the device of the invention has the advantage of facilitating their detection. By "DNA extraction" is meant any technique for isolating genomic or bacterial DNA from a biological sample, preferably from stool of a subject to be tested. In the context of this invention is meant a technique for isolating the DNA obtained from the stool of the subject. The DNA thus extracted may be genomic DNA or bacterial DNA. The DNA can then be used for molecular diagnostic purposes, in particular for the diagnosis of cancerous pathologies, such as colorectal cancers. There are different protocols for extracting DNA, which follow approximately the same basic scheme:
lyse des cellules, généralement à l'aide d'une solution de lyse, classiquement utilisée dans l'art antérieur et largement décrite, comme par exemple dans le cadre de la méthode dite d'extraction au phénol-chloroforme;  lysis of the cells, generally using a lysis solution, conventionally used in the prior art and widely described, such as in the context of the so-called phenol-chloroform extraction method;
élimination des protéines qui peuvent représenter un contaminant important pour l'étude de l'ADN génomique ou bactérien;  elimination of proteins that may represent a significant contaminant for the study of genomic or bacterial DNA;
élimination des autres acides nucléiques, notamment des ARNs;  elimination of other nucleic acids, especially RNAs;
concentration de l'ADN par précipitation à l'alcool absolu 100%, notamment a l'aide de plusieurs lavages à l'éthanol 70%.  concentration of the DNA by precipitation with absolute alcohol 100%, in particular using several washes with 70% ethanol.
L'homme du métier pourra se référer aux kits commerciaux, comme par exemple le kit QIAamp DNA Stool (Qiagen) et permettant une extraction facile, efficace et reproductible de l'ADN génomique.  Those skilled in the art may refer to commercial kits, such as the QIAamp DNA Stool kit (Qiagen) and allowing easy, efficient and reproducible extraction of genomic DNA.
Les solutions de lyse et d'extraction d'ADN permettent la mise en œuvre d'une extraction d'ADN d'un échantillon biologique, préférentiellement les selles du sujet à tester, selon les techniques classiquement décrites dans l'art antérieur. Cette extraction peut se faire par une première étape de lyse cellulaire. La lyse peut se faire de différentes manières: dislocation par voie mécanique, choc hypotonique, lyse chimique, ou encore digestion par protéinase K. Préférentiellement, cette lyse est une étape de lyse chimique. The DNA lysis and extraction solutions allow the implementation of a DNA extraction of a biological sample, preferably the stool of the test subject, according to the techniques conventionally described in the prior art. This extraction can be done by a first cell lysis step. Lysis can be done in different ways: mechanical dislocation, hypotonic shock, chemical lysis, or digestion with proteinase K. Preferably, this lysis is a chemical lysis step.
Dans un mode de réalisation particulier, la solution de lyse présente dans le dispositif de diagnostic selon l'invention est un mélange de détergent et de protéinaseK. Un tel mélange permet de dissocier les cellules, les tissus, les enveloppes des virus et de libérer les acides nucléiques. De manière non limitative, les détergents actuellement les plus usités sont le dodécyl sulfate de sodium (SDS), le sarcosyl, le Tween® 20, le Nonidet.  In a particular embodiment, the lysis solution present in the diagnostic device according to the invention is a mixture of detergent and proteinase K. Such a mixture makes it possible to dissociate the cells, the tissues, the envelopes of the viruses and to release the nucleic acids. In a nonlimiting manner, the currently most used detergents are sodium dodecyl sulfate (SDS), sarcosyl, Tween® 20, nonidet.
La présence de la solution de lyse au sein du dispositif selon l'invention est optionnelle et conditionne le procédé exploitant ledit dispositif. Aussi, si le dispositif ne comprend pas de solution de lyse, il conviendra de faire subir une étape de lyse cellulaire à l'échantillon biologique testé, avant de le mettre en contact avec ledit dispositif.  The presence of the lysis solution within the device according to the invention is optional and conditions the process using said device. Also, if the device does not include lysis solution, it will be appropriate to undergo a cell lysis step to the biological sample tested, before bringing it into contact with said device.
Après cette lyse cellulaire, il convient de procéder à l'extraction d'ADN génomique à proprement parler. Dans un mode de réalisation particulier, cette étape d'extraction se fait à l'aide d'un agent chaotropique, de manière préférée le thiocyanate de guanidium (GTC). Le GTC a pour fonction de disloquer les membranes des tissus de cellules et de dénaturer les protéines. En outre la suppression de ponts disulfures protéique, évitant toute renaturation protéique peut se faire par l'ajout de beta mercaptoéthanol dans le milieu. Aussi, dans un mode de réalisation particulier, la solution d'extraction d'ADN présent dans le dispositif de diagnostic selon l'invention est un mélange de thiocyanate de guanidium et de beta-2-mercaptoéthanol. After this cell lysis, genomic DNA extraction should be carried out as such. In a particular embodiment, this extraction step is carried out using a chaotropic agent, preferably guanidium thiocyanate (GTC). The function of CTG is to disrupt cell tissue membranes and denature proteins. In addition the deletion of protein disulfide bridges, avoiding protein renaturation can be done by the addition of beta mercaptoethanol in the medium. Also, in a particular embodiment, the DNA extraction solution present in the diagnostic device according to the invention is a mixture of guanidium thiocyanate and beta-2-mercaptoethanol.
La présente invention prévoit également la possibilité d'effectuer cette étape d'extraction selon la technique dite du phénol-chloroforme. Le phénol est en effet un puissant déprotéinisant. Son addition dans le milieu contenant le lysat cellulaire a pour conséquence de dénaturer les protéines. Après centrifugation, elles se situent à l'interface entre la phase aqueuse (qui contient les acides nucléiques) et la phase organique. Après transfert de la phase aqueuse dans un autre tube, les acides nucléiques sont traités par un mélange chloroforme-alcool isoamylique. Cette étape permet d'éliminer les traces de phénol. Cette étape est essentielle puisque le phénol est connu pour être toxique mais est également un inhibiteur de la Taq polymérase. Après mélange, et centrifugation et élimination de la phase organique, les acides nucléiques sont à l'état soluble dans la phase aqueuse.  The present invention also provides the possibility of carrying out this extraction step according to the so-called phenol-chloroform technique. Phenol is indeed a powerful deproteinizing. Its addition in the medium containing the cell lysate has the effect of denaturing the proteins. After centrifugation, they are at the interface between the aqueous phase (which contains the nucleic acids) and the organic phase. After transfer of the aqueous phase to another tube, the nucleic acids are treated with a chloroform-isoamyl alcohol mixture. This step eliminates traces of phenol. This step is essential since phenol is known to be toxic but is also an inhibitor of Taq polymerase. After mixing, and centrifugation and removal of the organic phase, the nucleic acids are in the soluble state in the aqueous phase.
Ainsi, la solution d'extraction d'ADN comprend préférentiellement du thiocyanate de guanidium et du béta-2-mercaptoéthanol. L'homme du métier est susceptible d'adapter la composition et les concentrations en thiocyanate de guanidium et de béta-2-mercaptoéthanol. L'homme du métier pourra se référer au manuel "Principes de biologie moléculaire en biologie clinique" par Amezian, Bogard et Lamoril, Campus Référence, Elsevier.  Thus, the DNA extraction solution preferably comprises guanidium thiocyanate and beta-2-mercaptoethanol. Those skilled in the art are able to adapt the composition and the concentrations of guanidium thiocyanate and beta-2-mercaptoethanol. Those skilled in the art can refer to the manual "Principles of molecular biology in clinical biology" by Amezian, Bogard and Lamoril, Campus Reference, Elsevier.
Typiquement, la solution d'extraction d'ADN comprend du thiocyanate de guanidium 4M et du béta-2-mercaptoéthanol à 0,5%.  Typically, the DNA extraction solution comprises 4M guanidium thiocyanate and 0.5% beta-2-mercaptoethanol.
Le dispositif de diagnostic selon l'invention comprend des billes, préférentiellement des billes de verre. Typiquement, il s'agit de billes de verre calibrées à 0,5 mm. Ces billes permettent d'améliorer, de manière mécanique, les performances de l'action chimique de la solution de lyse. Elles participent donc à la lyse des cellules présentes dans les selles du sujet à tester. The diagnostic device according to the invention comprises beads, preferably glass beads. Typically, it is glass beads calibrated at 0.5 mm. These beads make it possible to mechanically improve the performance of the chemical action of the lysis solution. They therefore participate in the lysis of the cells present in the stool of the test subject.
Préférentiellement, le volume total des billes n'excède pas 1/3 du volume total du dispositif selon l'invention.  Preferably, the total volume of the beads does not exceed 1/3 of the total volume of the device according to the invention.
Aussi, le dispositif de diagnostic selon l'invention est hautement avantageux car il permet de réunir trois approches de diagnostic du cancer du colon:  Also, the diagnostic device according to the invention is highly advantageous because it makes it possible to combine three approaches to diagnosing colon cancer:
la mesure du sang occulte;  the measurement of occult blood;
la mise en évidence des anomalies géniques d'ADN (mutation et méthylation); et  the identification of DNA gene abnormalities (mutation and methylation); and
la mise en évidence d'anomalies bactériennes fécales.  the detection of faecal bacterial abnormalities.
L'invention se rapporte en outre à une méthode de dépistage du cancer colorectal comprenant :  The invention further relates to a method of screening for colorectal cancer comprising:
A) la détermination de sang occulte dans les selles dudit sujet à l'aide du dispositif de diagnostic selon l'invention;  A) the determination of occult blood in the stool of said subject using the diagnostic device according to the invention;
B) l'extraction d'ADN génomique ou bactérien à partir des selles dudit sujet à l'aide du dispositif de diagnostic selon l'invention.  B) extraction of genomic or bacterial DNA from the stool of said subject using the diagnostic device according to the invention.
La détermination de sang occulte à l'étape A) de la méthode selon l'invention se fait à l'aide du test immunologique mis en œuvre grâce aux anticorps spécifiques de l'hémoglobine présents dans le dispositif de l'invention.  The occult blood determination in step A) of the method according to the invention is carried out by means of the immunological test carried out by means of the hemoglobin-specific antibodies present in the device of the invention.
Typiquement les selles du sujet à tester peuvent être mises au contact du dispositif directement après le prélèvement ou bien après une première étape de lyse cellulaire. Ce test immunologique révèle la présence de sang avec une grande précision. L'échantillon biologique est mis en contact desdits anticorps spécifiques de l'hémoglobine. La visualisation des réactions immunologiques peut être effectuée par tout moyen de détection, tels que des moyens directs ou indirects. Typically the stool of the subject to be tested can be brought into contact with the device directly after the sampling or after a first cell lysis step. This immunological test reveals the presence of blood with great precision. The biological sample is contacted with said hemoglobin-specific antibodies. Visualization of immunological reactions can be performed by any means of detection, such as direct or indirect means.
La détection directe peut se faire sans l'intermédiaire d'un marquage, on observe les réactions immunologiques par exemple par résonance plasmonique de surface ou par voltamétrie cyclique sur une électrode portant un polymère conducteur. De manière préférée, la détection directe se fait par l'intermédiaire d'un marquage dudit anticorps.  The direct detection can be done without the intermediary of a marking, the immunological reactions are observed for example by surface plasmon resonance or by cyclic voltammetry on an electrode carrying a conductive polymer. Preferably, the direct detection is via a labeling of said antibody.
Les systèmes indirects de détection peuvent être aussi utilisés, comme par exemple des ligands capables de réagir avec un anti-ligand. Les couples ligand/anti- ligand sont bien connus de l'homme du métier, comme par exemple le couple biotine/streptavidine. Dans ce cas, c'est le ligand qui porte le partenaire de liaison. L'anti-ligand peut être détectable directement par les réactifs marqueurs décrits au paragraphe précédent ou être lui-même détectable par un ligand/anti-ligand.  Indirect detection systems can also be used, for example ligands capable of reacting with an anti-ligand. The ligand / anti-ligand pairs are well known to those skilled in the art, such as the biotin / streptavidin pair. In this case, it is the ligand that carries the binding partner. The anti-ligand can be detectable directly by the labeling reagents described in the preceding paragraph or be itself detectable by a ligand / anti-ligand.
Ces systèmes indirects de détection peuvent conduire, dans certaines conditions, à une amplification du signal. Cette technique d'amplification du signal est bien connue de l'homme du métier.  These indirect detection systems can lead, under certain conditions, to amplification of the signal. This signal amplification technique is well known to those skilled in the art.
Typiquement, après conjugaison de l'hémoglobine, préférentiellement la globine, et de son anticorps spécifique, on procède à une purification, généralement mise en œuvre par chromatographie, par exemple d'exclusion, par affinité ou par dialyse.  Typically, after conjugation of the hemoglobin, preferably globin, and its specific antibody, purification is carried out, generally carried out by chromatography, for example exclusion, by affinity or by dialysis.
On peut ensuite évaluer la concentration en hémoglobine des selles analysées selon les techniques classiques de l'art antérieur. La présence de sang occulte dans les selles est corrélée avec un risque de présence d'une pathologie cancéreuse, telle qu'un cancer de l'intestin, du colon ou du rectum. The hemoglobin concentration of the stools analyzed can then be evaluated according to the conventional techniques of the prior art. The presence of occult blood in stool is correlated with a risk of presence of a cancerous pathology, such as cancer of the intestine, colon or rectum.
L'extraction d'ADN génomique ou bactérien à partir des selles dudit sujet à l'étape B) de la méthode se fait selon les modalités développées précédemment. The extraction of genomic or bacterial DNA from the stool of said subject in step B) of the method is carried out according to the modalities previously developed.
Dans un mode de réalisation particulier, la méthode de dépistage du cancer colorectal chez un sujet selon l'invention comprend en outre: In a particular embodiment, the method for detecting colorectal cancer in a subject according to the invention further comprises:
C) la détermination de la présence d'une mutation d'un gène marqueur du cancer colorectal; et/ou  C) determining the presence of a mutation of a marker gene for colorectal cancer; and or
D) la détermination d'une instabilité génétique au niveau des séquences répétées microsatellites de l'ADN choisies parmi Bat25 et Bat26; et /ou D) determining a genetic instability at the microsatellite repeat sequences of the DNA selected from Bat25 and Bat26; and or
E) la mesure du degré de méthylation d'au moins un des gènes choisi parmi NPY, PENK, Wifl et leurs fragments ou variants dans les selles dudit sujet à l'aide du dispositif selon l'une des revendications 1 à 4; et/ou. E) measuring the degree of methylation of at least one of the genes selected from NPY, PENK, Wifl and their fragments or variants in the stool of said subject using the device according to one of claims 1 to 4; and or.
F) l'identification et la quantification d'un profil bactérien fécal caractéristique de l'existence d'un cancer colorectal.  F) the identification and quantification of a faecal bacterial profile characteristic of the existence of colorectal cancer.
La méthode de l'invention prévoit une étape C) de détermination de la présence d'une mutation d'un gène marqueur du cancer colorectal. Préférentiellement, cette étape C) est une étape de détermination de la présence d'une mutation d'un gène choisi parmi le gène Kras et le gène APC. A l'aide de ses connaissances générales, l'homme du métier peut aisément déterminer la présence ou l'absence de mutations dans ces gènes dans l'échantillon biologique étudié à l'aide du dispositif selon l'invention. Une telle détermination de la présence ou l'absence de mutations dans ces gènes permet aux professionnels de la santé de déterminer dans quelle mesure un patient pourrait réagir à un traitement donné. The method of the invention provides a step C) of determining the presence of a mutation of a marker gene for colorectal cancer. Preferentially, this step C) is a step of determining the presence of a mutation of a gene selected from the Kras gene and the APC gene. With the help of his general knowledge, a person skilled in the art can easily determine the presence or absence of mutations in these genes in the biological sample studied using the device according to the invention. Such a determination of the presence or absence of mutations in these genes allows health professionals to determine how well a patient might respond to a given treatment.
Par "mutation", on entend tout phénomène de modification du matériel génétique. Ce terme englobe les insertions, les substitutions et les délétions.  By "mutation" is meant any phenomenon of modification of the genetic material. This term includes insertions, substitutions and deletions.
Par "gène Kras", on entend le gène codant pour la protéine Kras. De récentes études on montré que la présence d'une mutation du gène Kras est associée à une absence de bénéfice clinique aux traitements anti-EGFR. Ainsi, en déterminant la présence ou l'absence de mutation du gène Kras chez un sujet, les acteurs de santé peuvent adapter la thérapie adéquate pour ledit sujet. En effet, seuls les patients ayant une tumeur avec un gène Kras de type non-muté peuvent bénéficier d'un traitement anti-EGFR.  By "Kras gene" is meant the gene encoding the Kras protein. Recent studies have shown that the presence of a mutation in the Kras gene is associated with a lack of clinical benefit to anti-EGFR treatments. Thus, by determining the presence or absence of mutation of the Kras gene in a subject, health actors can adapt the appropriate therapy for the subject. Indeed, only patients with a tumor with a non-mutated Kras gene can benefit from anti-EGFR treatment.
Par "gène APC", on entend le gène codant pour la protéine " Adenomatous polyposis coli". Ce gène a été décrit comme possédant une activité suppressive de tumeurs. Aussi, il est actuellement reconnu qu'une mutation du gène APC chez un sujet donné, notamment une mutation conduisant à l'inactivation de ce gène, est associée à l'existence d'un cancer colorectal chez ledit sujet.  By "APC gene" is meant the gene coding for the protein "Adenomatous polyposis coli". This gene has been described as possessing tumor suppressor activity. Also, it is currently recognized that a mutation of the APC gene in a given subject, including a mutation leading to the inactivation of this gene, is associated with the existence of colorectal cancer in said subject.
La méthode de l'invention prévoit une étape D) de détermination d'une instabilité génétique au niveau des séquences répétées microsatellites de l'ADN choisies parmi Bat25 et Bat26. La fréquence des altérations génétiques des séquences répétées microsatellites de l'ADN peut être aisément déterminée par l'homme du métier. Par "Bat25" ou "Bat26", on désigne deux microsatellites quasi-homozygtes, quasi-monomorphes et mononucléotidiques. Bat25 correspond à une répétition de thymines (queue poly(T)) et Bat26 correspond à une répétition d'adénines (queue poly(A)). The method of the invention provides a step D) for determining a genetic instability at the level of the DNA microsatellite repeat sequences chosen from Bat25 and Bat26. The frequency of genetic alterations of the microsatellite repeat sequences of DNA can be readily determined by those skilled in the art. By "Bat25" or "Bat26", we designate two quasi-homozygous, quasi-monomorphic and mononucleotide microsatellites. Bat25 corresponds to a repetition of thymines (poly (T) tail) and Bat26 corresponds to a repeat of adenines (poly (A) tail).
Par "microsatellites", on entend de courtes séquences d'ADN formées de la répétition d'un motif lui-même constitué d'une à quatre bases. Par "instabilité génétique au niveau des séquences répétées microsatellites de l'ADN", se caractérise par la variation anormale du nombre de séquences répétées dans l'ADN tumoral comparé à l'ADN du même sujet provenant de tissu sain.  By "microsatellites" is meant short DNA sequences formed from the repetition of a motif consisting of one to four bases. By "genetic instability at the level of the repeated DNA microsatellite sequences", is characterized by the abnormal variation in the number of repeated sequences in the tumor DNA compared to the DNA of the same subject from healthy tissue.
La méthode de l'invention prévoit une étape E) de détection d'hyperméthylation de gènes marqueurs précédemment identifiés par les inventeurs et d'une manière générale de tout autre marqueur d'ADN humain ou bactérien spécifique des maladies digestives. Aussi, dans un mode de réalisation particulier, la méthode de dépistage du cancer colorectal selon l'invention comprend en outre la mesure du degré de méthylation d'au moins un des gènes choisi parmi Wifl, NPY, PENK et leurs fragments ou variants dans les selles dudit sujet à l'aide du dispositif selon les revendications. L'analyse de la méthylation de l'ADN peut être effectuée en utilisant des techniques basées sur la PCR qualitative et/ou quantitative comme la MSP (méthylation spécifique PCR), le séquençage bisulfite, la digestion par une enzyme de restriction sensible aux méthylations couplée avec la PCR. The method of the invention provides a step E) for detecting hypermethylation of marker genes previously identified by the inventors and in general of any other human or bacterial DNA marker specific for digestive diseases. Also, in a particular embodiment, the colorectal cancer screening method according to the invention further comprises measuring the degree of methylation of at least one of the genes selected from Wifl, NPY, PENK and their fragments or variants in the stool of said subject with the aid of the device according to the claims. DNA methylation analysis can be performed using qualitative and / or quantitative PCR-based techniques such as MSP (PCR specific methylation), bisulfite sequencing, methylation-sensitive restriction enzyme digestion coupled with PCR.
Par "méthylation" on entend l'addition d'un groupement méthyl au niveau du carbone 5' d'une cytosine dans un dinucléotide CpG. Ces dinucléotides sont peu fréquents dans la structure de l'ADN, sauf dans les "îlots" CpGs. Ces îlots sont typiquement représentés au niveau de la région promotrice des gènes. Ainsi, lorsqu'on parle de méthylation d'un gène, on fait référence à la méthylation de la région promotrice dudit gène. La présence d'un groupement méthyle en un site précis empêche l'interaction entre le gène et les facteurs de transcription. Typiquement, les groupes méthyles empêchent les facteurs de transcription de se fixer sur le site d'amplification et au promoteur, et à l'ARN polymérase de se fixer sur le site d'initiation. Aussi, la méthylation de la région promotrice a pour conséquence la répression de la transcription de l'ADN. L'expression "méthylation d'un gène" englobe la méthylation des îlots CpG de la séquence nucléotidique du gène mais également la méthylation des séquences nucléotidiques du promoteur du gène auquel cette expression s'applique. By "methylation" is meant the addition of a methyl group at the 5 'carbon of a cytosine in a CpG dinucleotide. These dinucleotides are infrequent in the structure of DNA, except in the CpGs "islets". These islets are typically represented at the level of the promoter region of the genes. Thus, when one speaks of methylation of a gene, reference is made to the methylation of the promoter region of said gene. The presence of a methyl group at a specific site prevents interaction between the gene and transcription factors. Typically, methyl groups prevent transcription factors from binding to the amplification site and promoter, and RNA polymerase from binding to the initiation site. Also, the methylation of the promoter region results in the repression of the transcription of the DNA. The term "methylation of a gene" encompasses the methylation of the CpG islets of the nucleotide sequence of the gene but also the methylation of the nucleotide sequences of the promoter of the gene to which this expression applies.
Par "PCR Multiplex", on entend une forme de PCR, généralement une PCR quantitative permettant l'amplification simultanée de plusieurs cibles d'intérêt en une seule étape, en utilisant une ou plusieurs amorces spécifiques. Cette technique est très avantageuse pour déterminer la présence de délétions, mutations, polymorphismes ou hyperméthylation de plusieurs marqueurs.  By "Multiplex PCR" is meant a form of PCR, generally a quantitative PCR allowing the simultaneous amplification of several targets of interest in a single step, using one or more specific primers. This technique is very advantageous for determining the presence of deletions, mutations, polymorphisms or hypermethylation of several markers.
Par opposition, l'expression "PCR Monoplex" fait référence à une forme de PCR, généralement une PCR quantitative, permettant l'amplification d'une seule cible d'intérêt.  In contrast, the term "Monoplex PCR" refers to a form of PCR, usually a quantitative PCR, allowing the amplification of a single target of interest.
Par "méthylation spécifique PCR", on entend une technique classique de l'art antérieur permettant de mesurer le degré de méthylation d'un gène. Cette technique se base sur le principe de la PCR quantitative. Typiquement, cette technique repose sur le traitement avec le bisulfite de sodium de l'échantillon d'ADN à étudier. Ce traitement permet de transformer chacune des cytosines non méthylées en uraciles dans l'ADN traité. L'échantillon ainsi traité subit ensuite une PCR avec des amorces spécifiques des gènes à traiter. La détermination de la nature des amorces spécifiques dépend de la séquence nucléotidique à amplifier. Dans le cadre de cette invention, la méthylation spécifique PCR est préférentiellement mise en œuvre en mode multiplex, on parle alors de Méthylation Spécifie PCR en mode multiplex, soit MSPM. By "PCR specific methylation" is meant a conventional technique of the prior art for measuring the degree of methylation of a gene. This technique is based on the principle of quantitative PCR. Typically, this technique relies on treatment with sodium bisulfite of the DNA sample to be studied. This treatment makes it possible to transform each of the unmethylated cytosines in uracils in the treated DNA. The sample thus treated then undergoes PCR with primers specific to the genes to be treated. The determination of the nature of the specific primers depends on the nucleotide sequence to be amplified. In the context of this invention, the specific PCR methylation is preferably carried out in multiplex mode, which is then referred to as PCR-specific methylation in multiplex mode, ie MSPM.
Par "gène Wifl", on entend le gène qui code pour la protéine WIF agissant comme inhibiteur de la voie Wnt. L'élément initiateur de la carcinogénèse colorectale est l'inactivation du gène APC dont la protéine joue un rôle relatif au contrôle de la prolifération, de l'apoptose et de la migration des cellules épithéliales intestinales. Cette inactivation provoque l'activation de certaines voies de signalisation, telles que celle de la WNT/Béta-caténine et la formation d'adénomes précoces. Il est connu que Wifl est l'un des gènes impliqués dans l'inhibition de la voie de signalisation Wnt. Aussi, l'hyperméthylation du gène Wifl s'accompagne de la levée d'inhibition de cette voie.  By "Wifl gene" is meant the gene that encodes the WIF protein acting as an inhibitor of the Wnt pathway. The initiating element of colorectal carcinogenesis is the inactivation of the APC gene whose protein plays a role in controlling the proliferation, apoptosis and migration of intestinal epithelial cells. This inactivation causes the activation of certain signaling pathways, such as that of WNT / beta-catenin and the formation of early adenomas. It is known that Wifl is one of the genes involved in the inhibition of the Wnt signaling pathway. Also, the hypermethylation of the Wifl gene is accompanied by the inhibition of inhibition of this pathway.
Par "gène NPY", on entend le gène qui code pour le neuropeptide Y ou By "NPY gene" is meant the gene which encodes neuropeptide Y or
NPY. NPY.
Par "gène PENK", on entend le gène qui code pour la neuropeptide proenképhaline A ou PENK.  By "PENK gene" is meant the gene which codes for the neuropeptide proenkephalin A or PENK.
Ces deux neuropeptides sont impliqués dans la prise alimentaire, l'absorption des nutriments et l'équilibre de la dépense énergétique de l'organisme. Ce sont des neuropeptides régulateurs des fonctions digestives qui peuvent également réguler la prise alimentaire et le comportement neuro-digestif. Par "fragment d'un gène", on entend une séquence dudit gène d'une longueur d'au moins 50 paires de bases, préférentiellement d'une longueur comprise entre 60 à 110 paires de bases. These two neuropeptides are involved in food intake, absorption of nutrients and the balance of energy expenditure of the body. They are neuropeptides regulating digestive functions that can also regulate food intake and neuro-digestive behavior. By "fragment of a gene" is meant a sequence of said gene having a length of at least 50 base pairs, preferably of a length of between 60 and 110 base pairs.
Par "pourcentage d'identité" entre deux séquences d'acides nucléiques ou au sens de la présente invention, on entend désigner un pourcentage de nucléotides entre les deux séquences à comparer, obtenu après le meilleur alignement, ce pourcentage étant purement statistique et les différences entre les deux séquences étant réparties au hasard et sur toute leur longueur. On entend désigner par "meilleur alignement" ou "alignement optimal", l'alignement pour lequel le pourcentage d'identité déterminé comme ci-après est le plus élevé. Les comparaisons de séquences entre deux séquences d'acides nucléiques sont traditionnellement réalisées en comparant ces séquences après les avoir alignées de manière optimale, ladite comparaison étant réalisée par segment ou par « fenêtre de comparaison » pour identifier et comparer les régions locales de similarité de séquence. L'alignement optimal des séquences pour la comparaison peut être réalisé, outre manuellement, au moyen de l'algorithme d'homologie locale de Smith et Waterman (1981), au moyen de l'algorithme d'homologie locale de Neddleman et Wunsch (1970), au moyen de la méthode de recherche de similarité de Pearson et Lipman (1988), au moyen de logiciels informatiques utilisant ces algorithmes (GAP, BESTFIT, BLAST P, BLAST N, FASTA et TFASTA dans le Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI). Afin d'obtenir l'alignement optimal, on utilise de préférence le programme BLAST, avec la matrice BLOSUM 62. Le pourcentage d'identité entre deux séquences d'acides nucléiques est déterminé en comparant ces deux séquences alignées de manière optimale, la séquence d'acides nucléiques à comparer pouvant comprendre des additions ou des délétions par rapport à la séquence de référence pour un alignement optimal entre ces deux séquences. Le pourcentage d'identité est calculé en déterminant le nombre de positions identiques pour lesquelles le nucléotide est identique entre les deux séquences, en divisant ce nombre de positions identiques par le nombre total de positions comparées et en multipliant le résultat obtenu par 100 pour obtenir le pourcentage d'identité entre ces deux séquences. "Percentage of identity" between two nucleic acid sequences or in the sense of the present invention, is meant to designate a percentage of nucleotides between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their entire length. By "best alignment" or "optimal alignment" is meant the alignment for which the percentage of identity determined as hereinafter is the highest. Sequence comparisons between two nucleic acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being performed by segment or by "comparison window" to identify and compare the local regions of sequence similarity. . The optimal alignment of the sequences for comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970) ), using the Pearson-Lipman (1988) similarity search method, using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI). In order to obtain the optimal alignment, the BLAST program is preferably used with the BLOSUM 62 matrix. The percentage identity between two nucleic acid sequences is determined by comparing these two optimally aligned sequences. nucleic acid sequence to be compared which may comprise additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide is identical between the two sequences, dividing this number of identical positions by the total number of positions compared and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
Par "variants présentant un pourcentage d'identité d'au moins 80 %, de préférence d'au moins 90 %, de façon plus préférée d'au moins 98 % ", on entend désigner les séquences nucléiques présentant, par rapport à la séquence nucléique de référence, certaines modifications comme en particulier une délétion, une troncation, un allongement, une fusion chimérique, et/ou une substitution, notamment ponctuelle, et dont la séquence nucléique présente au moins 80 , de préférence au moins 90 , de façon plus préférée au moins 98 , d'identité après alignement optimal avec la séquence nucléique de référence.  By "variants having an identity percentage of at least 80%, preferably at least 90%, more preferably at least 98%", are meant nucleic sequences having, relative to the sequence reference nuclei, certain modifications such as in particular deletion, truncation, elongation, chimeric fusion, and / or substitution, in particular punctual, and whose nucleic sequence has at least 80, preferably at least 90, more preferred at least 98, identity after optimal alignment with the reference nucleic sequence.
Cette étape E) de la méthode selon l'invention peut également comprendre la mesure du degré de méthylation d'un gène de ménage. Préférentiellement, cette étape de mesure du degré de méthylation du gène de ménage s'effectue de manière simultanée à la mesure du degré de méthylation d'au moins un des gènes choisi parmi Wifl, NPY, PENK et leurs fragments ou variants et/ou de manière simultanée à la mesure du degré de méthylation du gène Wifl ou un de ses fragments ou variants.  This step E) of the method according to the invention may also comprise the measurement of the degree of methylation of a household gene. Preferably, this step of measuring the degree of methylation of the household gene is carried out simultaneously to measure the degree of methylation of at least one of the genes selected from Wifl, NPY, PENK and their fragments or variants and / or simultaneously with measuring the degree of methylation of the Wif1 gene or a fragment or variant thereof.
Par "gène de ménage", ou "gène ménager" ou "gène constitutif", ou encore "housekeeping gene", on entend un gène constitutionnel qui code principalement pour des protéines essentielles aux fonctions cellulaires de base. De manière préférée, l'expression des gènes de ménage selon l'invention est ubiquitaire et stable entre les différents tissus. Préférentiellement le gène de ménage est choisi parmi les gènes codant pour l'albumine, la beta actine, l'alpha actine, beta microglobuline, préférentiellement l'albumine. Ces gènes de ménage présentent un intérêt majeur dans le contexte de l'invention. En effet, le degré de méthylation des gènes NPY, PENK et Wif 1 ou leurs fragments ou variants est exprimé par rapport à la méthylation d'un gène de ménage dont l'expression est stable. By "housekeeping gene", or "housekeeping gene" or "constitutive gene" or "housekeeping gene" is meant a constitutional gene that encodes mainly for proteins essential to basic cellular functions. Of preferred way, the expression of the housekeeping genes according to the invention is ubiquitous and stable between the different tissues. Preferably, the household gene is chosen from the genes encoding albumin, beta-actin, alpha-actin, beta-microglobulin, and preferably albumin. These household genes are of major interest in the context of the invention. Indeed, the degree of methylation of the NPY, PENK and Wif 1 genes or their fragments or variants is expressed with respect to the methylation of a household gene whose expression is stable.
Par "degré de méthylation" ou "taux de méthylation", on entend le pourcentage du nombre de sites CpG méthylées du gène cible par rapport à un standard. Par gène cible, on entend un gène pour lequel on veut mesurer le degré de méthylation. Dans le cadre de la présente invention, ces gènes cibles sont Wifl, NPY, PENK, l'albumine et leurs fragments ou variants. Par standard, on entend la séquence sauvage dudit gène cible ou bien la séquence d'un gène de ménage.  By "degree of methylation" or "level of methylation" is meant the percentage of the number of methylated CpG sites of the target gene compared to a standard. By target gene is meant a gene for which it is desired to measure the degree of methylation. In the context of the present invention, these target genes are WifI, NPY, PENK, albumin and their fragments or variants. By standard is meant the wild-type sequence of said target gene or the sequence of a household gene.
Le calcul du degré de méthylation se fait selon les techniques de quantification bien connues de l'homme du métier. Cette quantification peut être absolue ou relative. De manière préférée, son calcul se fait selon la technique dite du AACt bien connue de l'art antérieur. De manière préférée, la méthode de l'invention comprend en outre une étape de comparaison des degrés de méthylation d'une part des gènes Wifl et d'un gène de ménage, ou leurs fragments ou variants et d'autre part des gènes NPY et PENK ou leurs fragments ou variants à une valeur seuil, ladite valeur seuil étant déterminée en fonction des besoins en termes de spécificité et de sensibilité.  The degree of methylation is calculated according to the quantification techniques well known to those skilled in the art. This quantification can be absolute or relative. Preferably, its calculation is done according to the so-called AACt technique well known in the prior art. Preferably, the method of the invention also comprises a step of comparing the methylation levels on the one hand of the Wifl genes and of a household gene, or their fragments or variants and, on the other hand, of the NPY genes and PENK or their fragments or variants at a threshold value, said threshold value being determined according to the needs in terms of specificity and sensitivity.
Enfin, la méthode de l'invention prévoit une étape F) d'identification et la quantification d'un profil bactérien fécal caractéristique de l'existence d'un cancer colorectal. Préférentiellement, l'étape F) est une étape d'identification et de quantification d'un profil bactérien de bactéries appartenant aux genres Bacteroides ou Prevotella. Finally, the method of the invention provides a step F) for identifying and quantifying a faecal bacterial profile characteristic of the existence of cancer. Colorectal. Preferably, step F) is a step of identifying and quantifying a bacterial profile of bacteria belonging to the genera Bacteroides or Prevotella.
Il est aujourd'hui admis que la flore intestinale a une influence sur la santé et le bien être de l'individu. De récentes études ont mis en évidence l'ensemble des microorganismes constituant la flore intestinale, qui peut s'élever jusqu'à 500 types de microorganismes (Volker Mai et al, American Society for Nutritional Sciences, 2003; Qin et al., Nature, 2010; Lay et al., Applied and Environmental microbiology, 2005) composant 1014 bactéries/gr de selles. It is now accepted that the intestinal flora has an influence on the health and well-being of the individual. Recent studies have revealed all the microorganisms constituting the intestinal flora, which can be up to 500 types of microorganisms (Volker Mai et al, American Society for Nutritional Sciences, 2003, Qin et al. 2010; Lay et al., Applied and Environmental Microbiology, 2005) composing 10 14 bacteria / g of stool.
De récentes études mettent en lumière l'influence de cette flore intestinale dans le syndrome du colon irritable (Balfour Sartor, Gastroenterology, 2008), dans l'obésité (Bajzer et al., Nature, 2006), dans la maladie de Crohn (Seksik et al., Gut, 2003), et plus récemment dans le cancer.  Recent studies highlight the influence of this intestinal flora in irritable bowel syndrome (Balfour Sartor, Gastroenterology, 2008), in obesity (Bajzer et al., Nature, 2006), in Crohn's disease (Seksik et al., Gut, 2003), and more recently in cancer.
En effet, les inventeurs ont mis en évidence une modification de la flore colique de sujets atteints de cancers colorectaux, notamment une élévation de la quantité de bactéries appartenant aux genres Bacteroides ou Prevotella.  Indeed, the inventors have demonstrated a change in the colonic flora of subjects suffering from colorectal cancers, including an increase in the amount of bacteria belonging to the genera Bacteroides or Prevotella.
L'analyse de cet ADN bactérien, dans les conditions standards bien connues de l'homme du métier, permet donc la détermination de la composition de la flore intestinale du sujet à tester. Plus précisément, cette analyse permet à l'homme du métier de déterminer la quantité des bactéries cibles pertinentes pour le diagnostic du cancer colorectal dans les selles du sujet à tester.  The analysis of this bacterial DNA, under standard conditions well known to those skilled in the art, therefore allows the determination of the composition of the intestinal flora of the subject to be tested. More specifically, this analysis allows the skilled person to determine the amount of target bacteria relevant for the diagnosis of colorectal cancer in the stool of the subject to be tested.
Cette quantité peut ensuite être comparée à une valeur seuil, obtenue chez un sujet sain, c'est à dire ne souffrant pas de pathologies digestives, notamment de cancers colorectaux. EXEMPLE This quantity can then be compared to a threshold value, obtained in a healthy subject, ie not suffering from digestive pathologies, in particular colorectal cancers. EXAMPLE
Collecte de l'échantillon  Collection of the sample
Les selles fraîchement émises sont collectées dans un pot ou contenaire dédié. Le sujet doit mettre ses selles sur un support solide et à l'abri de toute contamination par eau ou urines.  Freshly made stools are collected in a pot or dedicated container. The subject should put his stool on a solid support and protected from any contamination by water or urine.
Mise en contact avec le dispositif selon l'invention  Contacting the device according to the invention
Le sujet doit tremper le piston du dispositif dans les selles ainsi recueillies et remettre le piston dans son étui. La quantité approximative de selles prélevées est estimée à 10 à 20 mg de selles diluées dans 2 mL de solution de lyse et d'extraction.  The subject must soak the piston of the device in the stool thus collected and put the piston back in its case. The approximate amount of stool removed is estimated to be 10 to 20 mg of stool diluted in 2 mL of lysis and extraction solution.
Détermination de la présence de sang occulte Determination of the presence of occult blood
Basé sur la réaction Ag-Ac, la présence de globine humaine est détectée et quantifiée in situ en cuve de lecture dédiée. La réaction immunologique Ag-AC est réalisée par la mise en contact de 50 μΐ de la solution d'anticorps à un volume de 50 μL· de solution biologique. Les deux solutions sont prélevées et introduites dans la cuve réactionnelle selon un mode manuel ou automatique. La lecture se fait de façon automatisée et directement au sein de ladite cuve de lecture selon le procédé d'opacité turbidimétrique: l'hémoglobine complexée à l'anticorps forme un agglomérat proportionnel à la concentration de globine. La lecture optique précisera le point correspondant sur une courbe d'étalon.  Based on the Ag-Ac reaction, the presence of human globin is detected and quantified in situ in dedicated reading tanks. The Ag-AC immunological reaction is carried out by contacting 50 μl of the antibody solution with a volume of 50 μl of biological solution. Both solutions are removed and introduced into the reaction tank in a manual or automatic mode. The reading is done automatically and directly within said reading tank according to the turbidimetric opacity process: the hemoglobin complexed with the antibody forms an agglomerate proportional to the concentration of globin. The optical reading will specify the corresponding point on a standard curve.
Extraction ADN génomique Genomic DNA Extraction
Protocole:  Protocol:
Après 48h de contact du complexe [selles (10-20 mg)- tampon de lyse cellulaire-billes] une extraction de l'ADN génomique a été effectuée en utilisant les principaux éléments constituant le kit QIAamp DNA Stool (Qiagen). Le protocole détaillé est décrit ci-dessous : After 48 hours of contact of the complex [stool (10-20 mg) - cell lysis buffer-beads] extraction of the genomic DNA was performed using the main components of the QIAamp DNA Stool Kit (Qiagen). The detailed protocol is described below:
1- les 2 ml du complexe [selles-tampon de lyse cellulaire-billes] sont récupérés en tube de 2 ml, puis vortexés en « Disruptor Beads » (Scientific Industries, Inc, USA) pendant 5 minutes et le tout centrifugé à 16,400 tr/min pendant 2 min  The 2 ml of the complex [stool-cell lysis buffer-beads] are recovered in 2 ml tube, then vortexed in "Disruptor Beads" (Scientific Industries, Inc., USA) for 5 minutes and all centrifuged at 16,400 tr / min for 2 min
2- prélever 1400 μΐ de surnageant et procéder au transfert du volume dans un tube de 2 ml  2- take 1400 μΐ of supernatant and transfer the volume to a 2 ml tube
3- ajouter une tablette InhibitEX (neutralise les inhibiteurs de PCR), vortexer pendant 1 min et laisser 1 min de contact à température ambiante  3- add an InhibitEX tablet (neutralize the PCR inhibitors), vortex for 1 min and leave 1 min contact at room temperature
4- centrifuger l'échantillon pendant 5 min à 16, 400 tr/min  4- centrifuge the sample for 5 min at 16,400 rpm
5- prélever le surnageant et l'introduire dans un tube de 1,5 ml puis centrifuger 3 min à 16, 400 tr/min  5- take the supernatant and introduce it into a 1.5 ml tube and centrifuge for 3 min at 16.400 rpm
6- introduire dans un tube de 2 ml, 600 μΐ de surnageant, 25 μΐ de protéinase K (100 mg/ml) et 600 μΐ de tampon AL. Vortexer le mélange pendant 15 secondes  6- Introduce into a 2 ml tube, 600 μΐ of supernatant, 25 μΐ of proteinase K (100 mg / ml) and 600 μΐ of buffer AL. Vortex the mixture for 15 seconds
7- incuber le mélange à 70°C pendant 10 min  7- Incubate the mixture at 70 ° C for 10 minutes
8- ajouter 600 μΐ d'éthanol 100% au lysat et vortexer  8- add 600 μl of 100% ethanol to the lysate and vortex
9- filtrer les 1800 μΐ de lysat dans une colonne QIAamp et centrifuger 1 min à 16,400 tr/min  9- Filter the 1800 μΐ of lysate in a QIAamp column and centrifuge for 1 min at 16.400 rpm
10- introduire 500 μΐ de tampon de lavage AW1 à la colonne et centrifuger 1 min à 16,400 tr/min  10- introduce 500 μΐ of washing buffer AW1 to the column and centrifuge for 1 minute at 16.400 rpm
11- introduire 500 μΐ de tampon de lavage AW2 à la colonne et centrifuger 3 min à 16,400 tr/min 12- installer la colonne sur un tube de 1,5 ml. Elution de l'ADN par 200 μΐ d'eau stérile 11- introduce 500 μΐ of washing buffer AW2 to the column and centrifuge for 3 min at 16.400 rpm 12- install the column on a 1.5 ml tube. Elution of the DNA with 200 μl of sterile water
13- détermination de la concentration de l'ADN par mesure spectrophotométrique à 260 nm  13- determination of the concentration of the DNA by spectrophotometric measurement at 260 nm

Claims

REVENDICATIONS
1. Dispositif de diagnostic comprenant : A diagnostic device comprising:
des anticorps spécifiques de l'hémoglobine, préférentiellement de la globine; optionnellement, une solution de lyse;  antibodies specific for hemoglobin, preferentially globin; optionally, a lysis solution;
une solution d'extraction d'ADN génomique et/ou bactérien; et  a genomic and / or bacterial DNA extraction solution; and
des billes de verre.  glass beads.
2. Dispositif selon la revendication 1 caractérisée en ce que la solution d'extraction d'ADN comprend du thiocyanate de guanidium et du béta-2-mercaptoéthanol. 2. Device according to claim 1 characterized in that the DNA extraction solution comprises guanidium thiocyanate and beta-2-mercaptoethanol.
3. Dispositif de diagnostic selon la revendication 1 ou 2, caractérisée en ce que les anticorps spécifique de l'hémoglobine, préférentiellement de la globine, sont des anticorps monoclonaux. 3. Diagnostic device according to claim 1 or 2, characterized in that the antibodies specific for hemoglobin, preferably globin, are monoclonal antibodies.
4. Dispositif selon l'une quelconque des revendications 1 à 3, caractérisée en ce que le volume de billes n'excède pas 1/3 du volume total dudit dispositif. 4. Device according to any one of claims 1 to 3, characterized in that the bead volume does not exceed 1/3 of the total volume of said device.
5. Méthode de dépistage du cancer colorectal chez un sujet, comprenant : 5. A method of screening for colorectal cancer in a subject, comprising:
A) la détermination de sang dans les selles dudit sujet à l'aide du dispositif de diagnostic selon l'une des revendications 1 à 4;  A) determining blood in the stool of said subject using the diagnostic device according to one of claims 1 to 4;
B) l'extraction d'ADN génomique ou bactérien à partir des selles dudit sujet à l'aide du dispositif de diagnostic selon l'une des revendications 1 à 4. B) extraction of genomic or bacterial DNA from the stool of said subject using the diagnostic device according to one of claims 1 to 4.
6. Méthode de dépistage du cancer colorectal chez un sujet selon la revendication 5, comprenant en outre: The method of screening for colorectal cancer in a subject of claim 5, further comprising:
C) la détermination de la présence d'une mutation d'un gène marqueur du cancer colorectal; et/ou  C) determining the presence of a mutation of a marker gene for colorectal cancer; and or
D) la détermination d'une instabilité génétique au niveau des séquences répétées microsatellites de l'ADN choisies parmi Bat25 et Bat26; et/ou E) la mesure du degré de méthylation d'au moins un des gènes choisi parmi Wifl, NPY, PENK, et leurs fragments ou variants dans les selles dudit sujet à l'aide du dispositif selon l'une des revendications 1 à 4; et/ou  D) determining a genetic instability at the microsatellite repeat sequences of the DNA selected from Bat25 and Bat26; and / or E) measuring the degree of methylation of at least one of the genes selected from Wifl, NPY, PENK, and their fragments or variants in the stool of said subject using the device according to one of claims 1 to 4; and or
F) l'identification et la quantification d'un profil bactérien fécal caractéristique de l'existence d'un cancer colorectal.  F) the identification and quantification of a faecal bacterial profile characteristic of the existence of colorectal cancer.
7. Méthode selon la revendication 6, caractérisée en ce que l'étape C) est une étape de détermination de la présence d'une mutation d'un gène choisi parmi le gène Kras et le gène APC. 7. Method according to claim 6, characterized in that step C) is a step of determining the presence of a mutation of a gene selected from the Kras gene and the APC gene.
8. Méthode selon la revendication 6 caractérisée en ce que l'étape F) est une étape d'identification et de quantification d'un profil bactérien de bactéries appartenant aux genres Bacteroides ou Prevotella. 8. The method of claim 6 characterized in that step F) is a step of identifying and quantifying a bacterial profile of bacteria belonging to the genera Bacteroides or Prevotella.
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