WO2009095840A1 - Procédé de détection simultanée de pathogènes et profilage génétique de l'hôte utilisant une matrice unique - Google Patents

Procédé de détection simultanée de pathogènes et profilage génétique de l'hôte utilisant une matrice unique Download PDF

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WO2009095840A1
WO2009095840A1 PCT/IB2009/050305 IB2009050305W WO2009095840A1 WO 2009095840 A1 WO2009095840 A1 WO 2009095840A1 IB 2009050305 W IB2009050305 W IB 2009050305W WO 2009095840 A1 WO2009095840 A1 WO 2009095840A1
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dna
host
pathogen
binding
disease
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Hendrik R. Stapert
Erik R. VOSSENAAR
Danielle Clout
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Koninklijke Philips Electronics N. V.
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    • 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
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a microarray device comprising probe oligonucleotides for detecting pathogen-DNA and host-DNA simultaneously.
  • the invention is further concerned with a method of detecting pathogen-0 DNA and host-DNA simultaneously comprising a step of preparing a liquid sample comprising pathogen-DNA and/or host-DNA followed by a step wherein the liquid sample is loaded into the chamber of the microarray device in order to contact the probe oligonucleotides of the microarray device, and finally a step of detecting interactions between the pathogen-DNA and/or host-DNA and said probe oligonucleotides.
  • the present invention is also concerned with a method of diagnosing an infectious disease and analyzing the genetic profile of the host at the same time to predict inter alia the reaction of the host to said disease and to applicate e.g. a tailormade regimen and/or to classify the risk group of the host. 0 BACKGROUND OF THE INVENTION
  • pathogen comprises a large number of different species such as for example bacteria, fungi and viruses with an even larger number of subtypes as for example Gram-negative, Gram-positive, anaerobic and aerobic bacteria.
  • pathogen comprises a large number of different species such as for example bacteria, fungi and viruses with an even larger number of subtypes as for example Gram-negative, Gram-positive, anaerobic and aerobic bacteria.
  • genetic material such as DNA. Said genetic material is unique for every single type of pathogen and known for many of said pathogens due to today's advanced sequencing technology. By using the pathogen's genomic information, the detection and identification of the pathogen(s) is reliable.
  • Sepsis is an immunologic response to infections and the involvement of genes relating to such an immune answer and a link of specific genetic markers within said genes or gene-regulating elements to susceptibility and prognosis has been demonstrated. It seems that there is a link between specific genetic markers of the immune system and the susceptibility of an individual carrying these markers to infectious diseases.
  • Balding et al. conclude that genetic variability in the IL-6, IL-IO and IL- IRN genes is associated with a poor outcome in meningococcal disease (Baldin et al., Genes and Immunity, 4, 2003, 533-540). In general, abnormal high cytokine blood levels (such as IL-6) are known to correlate with a bad prognosis for patients, as a multiple organ failure is often observed in such patients. Watanabe et al.
  • a microarray device comprising a chamber suitable for containing liquid samples wherein the liquid sample comprises host-DNA and/or pathogen-DNA. Furthermore, said microarray device comprises one or more different probe nucleotides which are positioned on different locations on a surface of the chamber wherein the probe nucleotides comprise probe nucleotides being capable of binding host-DNA and probe nucleotides being capable of binding pathogen-DNA.
  • the number of the different probe oligonucleotides for detecting pathogen-DNA is between 5 - 100; in said embodiment, the number of the different probe oligonucleotides for detecting host- DNA is between 5 - 100. In an even preferred embodiment, said number of different probe nucleotides for detecting pathogen-DNA is between 20 - 50, and said number of different probe oligonucleotides for detecting host-DNA is between 30 - 40.
  • the probe oligonucleotides of the microarray device being capable of binding pathogen-DNA comprise oligonucleotides which are capable of binding DNA of pathogens causing a specific disease.
  • the microarray device comprises probe oligonucleotides being capable of binding host-DNA which comprise oligonucleotides capable of binding host specific DNA factors.
  • Said host specific DNA- factors may be implicated in the susceptibility to said specific disease and/or prognosis of said specific disease.
  • said specific disease mentioned above comprises any infectious disease such as sepsis, influenza, pneumonia, meningitis, tuberculosis, malaria and HIV.
  • Said specific infectious disease comprises also any cancer caused by pathogens such as stomach cancer and cervical cancer.
  • the probe nucleotides of the microarray device according to the invention comprise 50 probe nucleotides being capable of binding pathogen-DNA selected from the group of pathogens causing sepsis. Furthermore, in this preferred embodiment of the invention, the probe nucleotides also comprise 36 nucleotides being capable of binding host-DNA selected from host specific DNA factors which are implicated in the susceptibility to sepsis.
  • the probe nucleotides of the microarray device according to the invention comprise probe nucleotides being capable of binding pathogen-DNA selected from the group of pathogens causing sepsis comprising Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus,
  • Enterococcus faecalis Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Streptococcus pneumoniae, Staphylococcus capitis, Klebsiella oxytoca, Streptococcus agalactiae, Proteus mirabilis, Staphylococcus cohnii, Staphylococcus haemolyticus, Acinetobacter baumannii, Enterococcus sp., Proteus vulgaris, Serratia marcescens, Staphylococcus warneri, Staphylococcus hominis, Streptococcus anginosus, Streptococcus mitis, Staphylococcus auricularis, Staphylococcus lentus, Streptococcus beta haem Group G, Streptococcus beta haem Group F, Streptococcus gordon
  • the probe nucleotides also comprise nucleotides being capable of binding host-DNA selected from the regions surrounding the positions of the genes as mentioned below of host specific DNA factors which are implicated in the susceptibility to sepsis comprising TNF ⁇ -308; IL6 -174, +1753, +2954; ILlO -1082, - 592; TLR2 -16934, +677, +753, +1349; TLR4 +299, +400; MyD88 -938, +1944; CD14 -159; TNFB +252; RIPK2 +283, +1027, +1039; TRIAD3 +479, +1032, +1292, +1303 as well as regions in IL8; IL18; IFN ⁇ ; Dectin-1 (CARD9); mannose receptor; DC-SIGN; MaI; Trif; Tram; Syk; Ticam;
  • the microarray device comprises probe nucleotides being capable of binding pathogen-DNA which are selected from nucleotides being capable of binding conserved regions of pathogen- DNA.
  • Said oligonucleotides may in this embodiment of the invention also be selected from nucleotides being capable of binding regions of the pathogen-DNA which are characteristic for the pathogenic potential to cause a disease.
  • the probe nucleotides of the microarray device being capable of binding host-DNA are selected from nucleotides being capable of binding host-DNA regions comprising insertions, deletions and SNPs which are characteristic for the susceptibility to a disease and/or prognosis of a disease.
  • the probe nucleotides of the microarray device being capable of binding pathogen-DNA additionally comprise probe nucleotides capable of binding pathogen-DNA regions which code for antibiotic resistancies.
  • the microarray device additionally comprises probe nucleotides being capable of binding host-DNA regions which are regulating the genes of drug-metabolizing enzymes and/or host-DNA regions which are coding for drug-metabolizing enzymes.
  • a method of detecting pathogen(s) and host specific factors simultaneously in a liquid sample comprises the steps of (a) Extracting DNA outside the human or animal body from a tissue sample
  • the DNA is amplified after said extraction step (step (a) of the method mentioned above) using a multiplex-PCR with primers specific for host-DNA and pathogen-DNA regions.
  • the present invention relates in one aspect to a method for diagnosing an infectious disease and predicting the host's reaction simultaneously. Said method comprises the following steps:
  • step (c) Assigning the disease according to the pathogen(s) detected in step (b)
  • the afore described method for diagnosing an infectious disease and predicting the host's reaction simultaneously comprises additionally in a preferred embodiment of the invention determining resistancies of the pathogen(s) in step (c). Said method comprises in a further preferred embodiment of the invention determining reactions of the host to drugs in step (d). The last step, step (e) in said two cases also additionally comprises in a preferred embodiment of the invention the diagnosis of said two parameters.
  • a microarray device as described above as well as the methods described above are used for developing a medicine tailormade for the individual patient.
  • Fig. 1 depicts the DNA sequence of the promoter region of the human TNF ⁇ -gene surrounding position -308 which is indicated by * (only sequence of the coding strand is given).
  • sequences of the primers used to amplify the region from position -21 A to -329 are indicated:
  • [»»»»»»»»»] indicates the "downstream- flanking" primer with the following sequence: 5 '-TCCCCAAAAGAAATGGAGG-S ' (Seq ID No. 1).
  • [ ⁇ « « « « «»] indicates the "upstream- flanking" primer with the following sequence: 5 '-CTTCTGGGCCACTGACTGAT-S ' (Seq ID No. 2).
  • probe nucleotides used for detection of the G/ A-SNP at position -308 are shown (see table below for nucleotide sequences of the probes used in the microarray).
  • sequences used in probes 1-8 are named after their corresponding starting position (ranging from -316 to -313) and the base at the position corresponding to position -308 of TNF ⁇ (G or A).
  • the probes themselves may contain further sequences such as po IyT -regions used for e.g. anchoring of the probes.
  • Fig. 2 A shows the design of a microarray used and the exact positions of the probes for detecting the G/ A-SNP at position -308 of TNF ⁇ .
  • Probes 1-8 are arranged in four different setups. Each setup as shown in Fig. 2 A is surrounded by a bold square. The positions of the control probes 9 and 10 in the array setup are also given.
  • probes 9 and 10 are identical.
  • Probe 10 is, however, printed at a lower concentration (4 times lower) than probe 10.
  • Fig. 2 B shows a hybridization result of a microarray with a design as depicted in Fig. 2 A after hybridization cycle 5.
  • the sample used for hybridization contains DNA which has been obtained from a patient being homozygous wildtype at position -308 of TNF ⁇ and which has been amplified using the primers depicted in Fig. 1.
  • Fig. 3 shows the corrected intensities of the probe-signals of porous microarrays used for the detection of the SNP at position -308 of TNF ⁇ against the hybridization cycle.
  • microarrays with identical probes 1-8 for sequences of these probes see figure 2 A) as indicated in the figure legends were used and the signals of these probes were detected upon each hybridization-cycle.
  • the signals of a "blanc" are also given for each microarray.
  • microarrays A-C were incubated with:
  • A a sample comprising DNA derived from a patient being homozygous wildtype at position -308 of TNF ⁇ ,
  • C a sample comprising DNA derived from a patient being heterozygous for the SNP at position -308 of TNF ⁇ .
  • the inventors have found that it is possible to analyze the genetic material of one or more pathogen(s) and genetic markers of the host simultaneously.
  • the simultaneous analysis leads to substantial information within short time which can be used as basis for further diagnostic and medical actions to be taken.
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates a deviation from the indicated numerical value of ⁇ 10 % and preferably ⁇ 5 %.
  • the present invention is based on the simultaneous detection of pathogens causing infections as well as host genetic markers for the susceptibility of the host towards the pathogen.
  • the invention relates in one embodiment to a microarray device comprising a chamber suitable for containing liquid samples (wherein the liquid sample comprises host-DNA and/or pathogen-DNA) and one or more different probe nucleotides which are positioned on different locations on a surface of the chamber wherein the probe nucleotides comprise probe nucleotides being capable of binding host-DNA and probe nucleotides being capable of binding pathogen- DNA.
  • probe nucleotide also referred to as “capture nucleotide” or “probe” is defined as a sequence of bases which comprises sequence regions complementary to either the leading or the lagging strand of the sequence subjected to analysis as well as other sequences important for structural reasons or anchoring the probe nucleotide on the microarray such as e.g. a polyT- sequence.
  • Said sequence subjected to analysis may be e.g. a genomic pathogen- or host- DNA region of interest. Because the "capture nucleotide” comprises sequences complementary to said region of interest, it is capable of binding said region of interest due to hybridisation with the complementary region.
  • hybridisation in the context of the present invention is used as known to the skilled person in the art, namely to describe the H-bonding interaction between the complementary bases A - T and G - C. Said hybridisation is of course dependent on the percent of identity between the sequences and the base sequence itself. As shown in the example of this invention, it is possible to analyse by such a system e.g. the binding of a sequence of interest to capture oligonucleotides differing in one base only (representing a "SNP” as defined below). This means that the sequence of interest will in this example only bind the capture nucleotide being fully complementary to its capture sequence.
  • probes based on the sequence information of e.g. pathogens or hosts such as humans or animals represents a routine method for the person skilled in the art.
  • probes are comprised of bases which are complementary to either the lagging or the leading strand of the corresponding DNA.
  • These complementary regions may have a length of 50, 40, 35, 30, 25, 20, 19, 18, 17, 16 or 15 bases. More preferably , the complementary regions are 14, 13, 12, 11, 10, 9, 8, 7 or 6 bases in length. Most preferred are complementary regions comprising 14 bases.
  • the DNA-regions the probes are complementary with may be chosen according to the diagnosis one would like to obtain.
  • the invention is concerned with a microarray device comprising probe nucleotides being capable of binding host-DNA and probe nucleotides being capable of binding pathogen-DNA.
  • probes being capable of binding pathogen-DNA will be described as well as pathogens and some examples of diseases caused by pathogens.
  • the probes would be designed such that they detect different pathogens known to be the "usual suspects", i.e. pathogens which are most prominent and known to be aggressive.
  • the strategy of probe-design may be different.
  • the pathogens would be classified according to a disease they cause or according to the symptoms of a disease.
  • the probes would thus cover a certain area of an infectious disease, as for example sepsis or any other disease mentioned below by being complementary to DNA-regions of pathogens causing said sepsis or disease.
  • This may also comprise further defining the sub-types of pathogens to clearly identify them (e.g. the type of Hepatitis).
  • It is furthermore possible to screen for the presence of a certain type of pathogens by using a DNA-region which is common to all of them (to identify e.g. the presence of streptococci). By using a DNA-region which is only present in one specific subtype, it is further possible to identify said specific subtype (e.g. streptococcus pneumonia).
  • probes may be designed to bind to and thereby identify regions in the pathogen-DNA which correspond to e.g. the aggressiveness of the pathogen.
  • An example for this is the acquired immunity towards antibiotics which many pathogens show today. This immunity is based on either plasmid or genome-integrated DNA-sequences which code for enzymes or regulate the expression of said enzymes which are capable of transferring said immunity to the pathogen e.g. by degrading the antibiotic drug or metabolizing it into an uneffective compound.
  • Other factors may contribute to the characteristic pathogenity as for example insertions as repeats or single nucleotide polymorphisms in promoters of certain pathogenic genes leading e.g. to a higher expression rate of said genes.
  • probes specifically designed to bind to complementary DNA- regions of pathogenic DNA it is possible to detect, identify and characterize the pathogen(s). This approach is largely based on research carried out to describe pathogens and their characteristics by relying on their corresponding genomic material and also unique fingerprints found in this genomic material.
  • pathogen in the context of the present invention comprises any pathogen capable of invading the host and causing an infectious disease.
  • pathogen as defined according to the invention may be a virus or a microorganism comprising bacteria, protozoa, fungi and parasites (in case of parasites e.g. different worm species such as a roundworm or a tapeworm). Due to the design of the invention, any proteinaceous pathogens such as prions do not fall under the present definition of a "pathogen” as they lack genomic material.
  • infection is caused by pathogens which are invading the body. The invasion may be limited to a particular body region or may be widespread in the bloodstream.
  • infection is typically understood as the inflammatory response to the presence of pathogens and/or invasion of normally sterile tissue by pathogens.
  • Bacteremia defines the presence of viable pathogens in the blood.
  • infectious disease also comprises any form of cancer which is caused by pathogens.
  • the above-mentioned inflammatory response is usually the first step of the body's own immune system which is felt by the host and which may be diagnosed.
  • SIRS systemic inflammatory response syndrome
  • severe sepsis relates to a sepsis as defined above in combination with organ dysfunction such as cardiovascular, renal, respiratory or hepatic failure and hypotension or hypotension.
  • organ dysfunction such as cardiovascular, renal, respiratory or hepatic failure and hypotension or hypotension.
  • the manifestations of hypoperfusion may include lactic acidosis, oliguria, acute alteration in mental status etc.
  • a "septic shock” may occur. This is defined as a persistent hypotension despite adequate fluid resuscitation accompanied by signs of hypoperfusion or organ dysfunction.
  • bacteria comprise E. coli causing urinary tract infections, Mycobacterium tuberculosis causing tuberculosis, Bacillus anthracis causing anthrax, Salmonella causing foodborne illness, Staphylococcus aureus causing toxic shock syndrome, Streptococcus pneumoniae causing pneumonia, Streptococcus pyogenes causing strep throat, Heliobacter pylori causing stomach ulcers as well as stomach cancer, Treponema pallidum causing syphilis, Bordetella pertussis causing pertussis, Clostridium tetani causing tetanus, and Francisella tularensis causing tularemia.
  • viruses comprise Hepatitis A, B, C, D and E viruses causing liver diseases, Influenza virus causing flu, Herpex simplex virus causing herpes, Molluscum contagiosum causing rash, Human Immunodeficiency virus causing AIDS, Poliovirus causing poliomyelitis, measles virus causing measles and Human Papilloma Virus causing cervical cancer.
  • protozoa examples comprise Cryptosporidium causing cryptosporidiosis, Giardia lamblia causing giardiasis, Plasmodium causing malaria and Trypanosoma cruzi causing chagas disease.
  • fungi comprise Pneumocystis jirovecii causing opportunistic pneumonia, Tinea causing ringworm, Candida causing candidiasis, Histoplasma capsolatum causing histoplasmosis and Cryptococcus neoformans causing cryptococcis.
  • infectious diseases or body regions often targeted by infectious diseases and the causative pathogens are listed.
  • infectious diseases or body regions often targeted by infectious diseases and the causative pathogens are listed.
  • the devices according to the invention may comprise a plurality of probes being able of detecting a plurality of pathogens, as discussed hereinafter. Diseases listed below may be diagnosed by the present invention by detecting several pathogens which are able to cause infectious diseases and/or accompany said diseases.
  • Sepsis is a disease caused by different pathogens.
  • the terms "sepsis”, “severe sepsis” and “septic shock” have been defined above. They represent different stages of severity of the body' response to an infection. Of course, they cannot be regarded as defined steps but rather as a continuum between the various possible stages of a response to an infection.
  • most prominent pathogens capable of causing sepsis and the more severe stages of sepsis may be selected from the following group comprising in a general listing Gram-negative and Gram-positive bacteria, polymicrobials, anaerobes and fungi and in a more specific listing Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Enter ococcus fiaecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Streptococcus pneumoniae, Staphylococcus capitis, Klebsiella oxytoca, Streptococcus agalactiae, Proteus mirabilis, Staphylococcus cohnii, Staphylococcus haemolyticus, Acinetobacter baumannii, Enterococcus sp., Proteus vulgaris, Serratia marcescens,
  • Pasteurella multocida Propionibacterium acnes, Ralstonia pickettii, Salmonella ser. Paratyphi B and Yersinia enterocditi.
  • Pneumonia is a also a disease caused by different pathogens.
  • Most prominent pathogens capable of causing pneumonia may be selected from the following group comprising MRSA, Staph, aureus, Pseudomonas sp., Haemophilus sp., Streptococcus pneumoniae, Streptococcus sp., Klebsiella sp., Escherichia sp., Enterobacter sp., Acinetobacter, Mycobacterium tuberculosis, Aspergillus sp., Chlamydia pneumoniae, Mycoplasma pneumoniae, Herpes simplex virus, Cytomegalovirus, Adenovirus ter sp., Candida sp., Serratia sp., Proteus sp., Legionella sp., Listeria monocytogenes, Stenotrophomonas sp., Morganella sp., Moraxella sp. and
  • meningitis Yet another disease caused by different pathogens is meningitis.
  • Most prominent pathogens capable of causing meningitis may be selected from the following group comprising Enterovirus, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, MRSA, Mycobacterium tuberculosis, Staphylococcus epidermidis (& CNS in general), Group B Strep, Toxoplasma gondii, Herpes simplex virus I, Herpes simplex virus II, Cryptococcus neoformans, Cytomegalovirus, Varicella zoster virus, Treponema pallidum, Pseudomonas aeruginosa, E.coli, Enterobacter aerogenes, Proteus morganii, Klebsiella pneumoniae, Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida
  • Infectious diseases of the respiratory tract may be caused by Haemophilus influenzae, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia trachomatis, Bordetella pertussis, Streptococcus pneumoniae, Chlamydophila pneumoniae, Human adenovirus, Human respiratory syncytial virus, Human parainfluenza virus I and 3, Influenza A virus and Influenza B virus.
  • Diarrhoeal infectious diseases may for example be caused by Vibrio cholerae, Shigella dysenteriae, Salmonella typhi, E. coli, Campylobacter, Clostridium difficile, Listeria monocytogenes, Salmonella eneritidis, Norwalk virus, rotavirus, Cryptosporidium parvum, Cyclospora cayetanensis, Giardia inestinalis, Encephalitozoon inestinalis and Entamoeba histolytica.
  • Trypanosomiasis may be caused by several pathogens, such as Tryposoma brucei gambiense or Trypanosoma brucei rhodesiense.
  • Otitis media may be caused by Haemophilus influenza, Streptococcus pneumonia, Streptococcus pyogenes or Moraxella cararrhalis.
  • Leishmaniasis is another example for a disease wherein several pathogens are implicated. These pathogens may be selected from the group of Leishmania tropica, Leishmania donovani, Leishmania mexicana venezuelensis, Leishmania garnhami, Leishmania pifanoi, Leishmania braziliensis, Leishmania peruviana, Leishmania colombiensis, Leishmania lainsoni, Leishmaniashawi, Leishmanianaiffi, Leishmania guyanensis, Leishmania panamensis and Leishmania chagasi.
  • the above list is meant to be an overview and not as an exclusive list of pathogens and infectious diseases which can be detected according to the invention.
  • pathogens causing infectious disease may also be detected by the present invention as long as a probe can be designed being capable of binding to the pathogen-DNA.
  • subtypes of most of the pathogens listed here which may be classified e.g. according to their infectious potential. These subtypes are also not mentioned explicit, but may easily be detected by the present invention (e.g. by a sequence comparison to identify differences on a DNA-level and design of the corresponding probes capable of binding these specific DNA-regions) and are, therefore, within the scope of the invention.
  • different Human Papilloma Virus- subtypes are associated with different developments of benign or malignat lesions and, therefore, with different severity of the disease.
  • pathogens may be detected on a microarray device according to the invention on which the probes being capable of binding pathogen-DNA are designed accordingly.
  • a patient may be an immunocompromised patient or a patient receiving drugs weakening the general state of the immune system, such as a chemotherapy.
  • Said pathogens may be selected from pathogens known to cause secondary infections.
  • said pathogens may be selected e.g.
  • VZV Via Zoster Virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr Virus
  • Mycobacterium tuberculosis Legionella spp., Listeria monocytogenes, Salmonella typhi, Candida spp., Aspergillus spp., Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis carinii, Toxoplasma gondii, Cryptosporidia, Leishmania spp. and C difficile.
  • resistance-genes (which are also meant to comprise plasmid-DNA) may be analyzed in order to determine the resistance to drugs selected from the group comprising Methicillin, Metronidazole as well as Vancomycin.
  • Said “resistance-genes” may be selected from the group comprising MecA for analysis of a Methicillin-resistance; Van A, Van B, Van C for analysis of a Vancomycin-resistance, TEM (coding for ESBL [extended spectrum beta lactamases]) for analysis of resistance to ESBL as well as KPC (plasmid-mediated carbapenemase).
  • pathogens within the meaning of the present invention and including the examples listed above may be detected, identified and genetically characterized by capture oligonucleotides which are specifically designed for this purpose.
  • the genomes of many pathogens are sequenced and said sequence information is accessible.
  • the analysis of the host's genome may be used to predict the corresponding reaction of the host to the infection and/or disease and may help to find the best and most effective treatment or observation strategy.
  • a combination of the pathogen-DNA and the host-DNA may lead to very profound information onto which individual therapy may be based. It has been mentioned that certain genetic signatures of the host allow a prediction of the host's reaction to an infection.
  • the genetic signatures as defined below might be involved in the regulation of genes or genes themselves coding for factors implicated in the immune answer or in the general reaction to an infection and/or a drug and/or in the overall constitution of the host. These aspects are in general mainly referred to as the susceptibility of the host to an infection. In case the genetic signature shows a higher "susceptibility" to a certain disease, the patient should be observed more closely and some further medical steps may be initiated. Also, the prognosis of the disease in the host may be predicted by interpreting genetic signatures. Furthermore, it is possible to analyze how a patient will likely respond to certain treatments (e.g. by analyzing the reaction of said patient to a medicament based on the genetic signatures of e.g. drug-metabolizing enzymes).
  • genetic signature which is also referred to as "host specific factor(s)" is meant to encompass certain regions in the host-DNA.
  • host specific factor(s) For said DNA regions in the host, different conditions are known correlating to different outcomes. "Different conditions” in this case refers mainly to differences in the DNA compared to a wildtype situation. These differences in the DNA compared to a wildtype situation are the “genetic signatures" which may correlate with host specific reactions to a certain disease and thus the susceptibility.
  • Such host specific factors may be characterized by insertions, deletions and single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • “Insertions” represent regions in the DNA wherein a DNA-sequence comprising one base or several bases has been integrated at a position in the genomic DNA where in the wildtype such a sequence is not present.
  • the inserted sequences may be as short as one or a few bases ranging to large regions of e.g. tandem repeats inserted into the genome.
  • an insertion of a single base may have a dramatic effect on the gene as it may lead to a frame-shift.
  • DNA-regions carrying "deletions” are characterized by a genotype wherein one or several bases are missing compared to the wildtype. As for the insertions, this may range from one or a few bases to large regions. Again, this affects genes or gene-regulating sequences, as important information may be vanished due to the deletion.
  • a single nucleotide polymorphism is defined as a change in a single base of the genome differing from the wildtype base at that position. Furthermore, the prevailing SNP definition indicates that an inherited allelic variation must have > 1% population frequency in order to be classified as a SNP. For example, the -308 position of the TNF ⁇ -gene, which is in the promoter region of said gene, is a G for the wildtype. There is, however, in certain cases an A detected at this position. In case both alleles carry the wildtype G, the host is homozygous wildtype at position - 308 of the TNF ⁇ -gene.
  • the host is homozygous for the SNP at position -308 of the TNF ⁇ -gene. If the two alleles differ with respect to that position (an A in one allele a B in the other allele), the host is heterozygous.
  • the three-dimensional structure of a protein is defined by its amino acid sequence and certain amino acids on specific position, respectively.
  • the structure in turn is very important for the function of said protein, be it e.g. a structural, an enzymatic function, the possibility to dimerize and so on.
  • the structure of the protein may be compromised at least partly or it may be misfolded in general. Thereby, it may be not functional any more. The same applies even more if some sequences are deleted or insertions are present which disrupt the overall structure.
  • NEMO is implicated in the regulatory system of NF- ⁇ B-expression. NEMO is located either within the nucleus or cytoplasm. This localization is dependent on the modification by a small-ubiquitin-like modifier called SUMO. Furthermore, NEMO is also target of ubiquitylation itself, leading to its degradation. These modification always target lysine- residues of the protein. In case of a SNP in the NEMO-gene leading to a base exchange in the sequence coding for one of these lysines which are targets for modification, the resulting amino acid may be different than lysine.
  • regulatory proteins are comprised of a large protein-complex with many interactions among the proteins. Also, the interactions between the DNA-sequences of promoters or further upstream or downstream gene -regulatory elements and regulatory proteins are crucial for gene-expression. If a SNP is present at a pronounced base important for said interaction, this might have a severe impact on the whole regulation process of the corresponding gene expression. Even more pronounced is the effect of an insertion: If this insertion has a very positive effect on the gene expression, it will lead to enhanced expression. The contrary might be true for a deletion: no complexes may be able to form at all in the promoter region and thereby the gene might be silenced.
  • any host-specific factors in genes and/or gene-regulating sequences associated or connected with the immune system is of special interest for designing the corresponding probes.
  • This comprises e.g. Toll-like receptors such as TLRl, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11, downstream factors such as NF-KB or MAPKs, pro -inflammatory compounds and inflammatory compounds such as interleukins (ILs), TNF ⁇ and so on.
  • PRRs pattern-recognition receptors
  • ILs such as IL-6 and IL-10.
  • the SNP at position -1082 of the IL- 10 gene is e.g. in the promoter region of IL-10.
  • probes for detection as mentioned in the following may be combined (probes for the "the host-specific factors"), namely for detecting SNPs at TNF ⁇ -308; IL6 -174, +1753, +2954; ILlO -1082, -592; TLR2 - 16934, +677, +753, +1349; TLR4 +299, +400; MyD88 -938, +1944; CD14 -159; TNFB +252; RIPK2 +283, +1027, +1039; TRIAD3 +479, +1032, +1292, +1303 as well as SNPs and/or deletions and/or insertions in regions of the following
  • probes detecting host specific factors like drug metabolizing enzymes or other factor important for the classification and characterization of the host with respect to the effectiveness of drugs and/or any adverse reaction(s) to drugs is carried out following standard protocols based on the corresponding DNA-sequences which should be used.
  • the design of probes being capable of binding host-DNA is identical.
  • probes for detecting host-specific factors such as insertions, deletions or SNPs
  • only knowledge of certain genetic markers is needed.
  • the insertion, deletion or SNP may be detected by probes being capable of binding this specific setup only.
  • the wildtype situation may also be covered by probes being capable of detecting the wildtype sequence.
  • the corresponding base may preferably not be located on the end of such a probe as this might influence the hybridisation and therefore, the result. Most preferably, this base is positioned in the center of said probe. As shown for the probes in the table for Fig. 2 A (in the description of the figures), however, the corresponding base may be located at position six from the end of the probe being composed of 14 complementary nucleotides in total.
  • the probe may be designed such that the "ends" flanking in the wildtype situation the "deleted sequence" which are linked in the deletion-case may be used as sequence covering both of these flanking regions by e.g. comprising seven bases of each of said region. In the wildtype case, the "deletion" is present and, therefore, no hybridisation will take place with a probe designed as set out above. Finally, an insertion may be detected by chosing DNA-regions of the inserted sequence and the flanking wildtype sequence. Therefore, part of the wildtype- sequence (e.g. seven bases) flanking the insertion and part of the insertion sequence
  • probe for detecting host-specific factors (e.g. the following seven bases after the chosen flanking region) may be used to design a corresponding probe.
  • the design of probe for detecting host-specific factors represents in most cases a routine technique for the skilled person in the art.
  • one set of probes being capable of binding pathogenic-DNA is present on one part of the microarray device. Thereby, pathogens as well as corresponding diseases may be identified.
  • Another set of probes being capable of binding host-DNA is present on the same microarray and allows the determination of genetic factors of the host such as susceptibility to a certain disease.
  • probes for detecting e.g. SNPs, deletions, insertion as well as complementary regions may be combined in any way to provide for the final goal of diagnosing a pathogen/ disease as well as analyzing host- specific factors.
  • the two sets of probes may be chosen such that they provide information in combination, i.e. the "pathogen-set” may be designed to detect and analyze certain pathogens causing sepsis with a corresponding "host-set” detecting host-specific factors which are implicated in sepsis and/or the general immune answer.
  • Another possible setup is the detection of pathogens causing pneumonia in combination with host-specific factors implicated in pneumonia and/or the general immune answer.
  • a possible setup is the detection of pathogens causing meningitis in combination with host-specific factors implicated in pneumonia and/or the general immune answer.
  • Another possible setup is the detection of pathogens in immunocompromised patients in combination with host-specific factors implicated in the general immune answer.
  • Yet another possible setup is the detection of pathogens causing influenza (such as Haemophilus influenza) in combination with host-specific factors implicated in influenza and/or the general immune answer.
  • pathogens causing cervical cancer such as HPVs
  • pathogens causing cervical cancer such as HPVs
  • stomach cancer such as Heliobacter pylori
  • host-specific factors implicated in stomach cancer and/or the general immune answer.
  • the number of probes present on a surface of the Microarray may be in total either about 500, 400, 350, 340, 330, 320, 310, 300, 280, 250, 240, 230, 220, 200, 180, 150, 140, 130, 125, 120 or 110. More preferred is a number of about 105, 104, 103, 102, 101, 100, 98, 95, 90, 86, 85, 84, 80, 70, 60, 50 , 40, 30 or 20.
  • the total number comprises probes for detection of pathogen-DNA as well as probes for detecting host-DNA.
  • the number of probes being capable of binding pathogen- DNA may be about 110, 90, 80, 70, 60, 50, 40, 30, 20 or 10.
  • said number may be between 5 and 100.
  • the number of probes being capable of binding host-DNA may be about 50, 45, 40, 36, 35, 30, 25, 20, 15 or 10. Preferably, it may be between 20 and 30.
  • a density ⁇ 400 dots/ cm 2 may be used.
  • a "dot" defines a plurality of identical probes at a given local concentration at this spot. However, the density may also be different due to the techniques of probe-printing and number of probes needed for the corresponding analysis.
  • the term "chamber" of the array defines a closed room wherein the probes are arranged such that their capture/ binding/ hybridizing oligonucleotides parts are presented into the defined room such that they are able to contact other molecules present in said room.
  • said room/ chamber may be open for a certain step during the experimental procedure such as the loading of a liquid sample into said room.
  • the chamber may be closed to account for a closed reaction system with defined reaction parameters such as the concentrations of certain molecules within said chamber.
  • sample refers to any liquid which is to be analyzed by using the invention. Said sample is supposed to comprise one or more species of DNA to be detected. Of course, the sample may comprise further one or more additional agents such as diluents, solvents or buffers that may result from optional purification and/or processing steps of the sample prior to the analysis.
  • blood of the host comprising host-DNA and optional pathogen-DNA in case of an infection represents such a sample.
  • the blood sample may be subject to further experimental procedures such as described below in order to extract DNA. However, other protocols leading to the DNA of said sample in a condition wherein it may be analyzed by the device according to the invention may also be used.
  • DNA microarrays used for the present invention may comprise many different setups with regard to e.g. the surfaces and/or supports of said arrays. These may be glass slides (such as known from i.a. Affymetrix, Agilent, Nimblegen, CodeLink), microbeads (such as known from i.a. Illumina) or porous membranes (such as known from i.a. Clondiag, Xceed, Pamgene, Philips). With regard to the setup and design of the probes used for the microarray of the present invention, however, the design described above comprising probes capable of binding pathogen-DNA and probes capable of binding host-DNA is employed.
  • the present invention also discloses a method of detecting pathogens and host-specific factors simultaneously.
  • the invention in one embodiment relates to a method of detecting pathogen(s) and host specific factors simultaneously in a liquid sample.
  • Said method comprises the steps of a) Extracting DNA outside the human or animal body from a tissue sample b) Solubilizing said DNA to obtain a liquid sample comprising host-DNA and/or pathogen-DNA c) Loading the liquid sample into the chamber of the microarray device according to the invention in order to contact the probe oligonucleotides of said microarray device with the liquid sample d) Detecting interactions between the host-DNA and/or the pathogen-DNA comprised in the liquid sample and the probe nucleotides.
  • Both, the DNA of the host-specific factors as well as the DNA of the pathogen(s) may be comprised in a liquid sample. How such a sample may be obtained is described further below where the method of diagnosing an infectious disease and analyzing the genetic profile of the host simultaneously is described.
  • the DNA comprising host and in case of an infection also pathogen-DNA
  • the DNA is extracted. This may again be done by any standard method known to the skilled person in the art and may comprise steps of cell-disruption, centrifugation in order to separate the nuclei from other cell organelles, disruption of said nuclei as well as the precipitation of DNA by e.g. high concentrations of salt.
  • any other methods may be used.
  • the obtained DNA may then be solubilized in a suitable amount of a suitable buffer and used directly as liquid sample for the method of detecting mentioned below.
  • an amplification step may optionally be done first: To do said amplification, the precipitated DNA may be solubilized in a suitable amount of a suitable buffer in order to perform a PCR using primers designed to amplify the DNA-regions which are detected by the probes present in the microarray according to the invention. Therefore, the amplification step is dependent on the probes used in the microarray-setup, as such DNA-regions need to be amplified which are subjected to detection later on. As PCR-method, a multiplex-and/or multichamber PCR may be carried out. Except from the primer-design, however, this amplification step represents a standard method for the skilled person in the art and may be performed by various techniques.
  • the primers may be designed such that the amplification may in a preferred embodiment be carried out in only one step. In an also preferred embodiment, said amplification is carried out in two steps. After completion of this amplification, the DNA may be isolated and solubilized again in order to get rid of compounds such as enzymes that might disturb the hybridisation step. After said amplification, however, the DNA is also comprised in a liquid sample for the further steps of detection.
  • the DNA may also be labelled using e.g. fluorescently labelled primers in order to facilitate the detection step for interactions with the probes as described in detail below.
  • fluorescent labels may be used: Cy-dyes like Cy-3 or Cy-5, FITC, Alexa-Red, fluorescein, rhodamin, lanthanides and so on.
  • said liquid sample may in the next step be loaded into the chamber of the microarray device as described above. This step may e.g. be done by pipetting the sample into said chamber followed by sealing the chamber. Alternatively, the liquid sample may be loaded through a water-impermeable membrane of the microarray device by using a syringe.
  • the DNA comprised in the liquid sample may now be contacted with the probes of the microarray device of the invention as described above in order to identify the (optionally) amplified DNA by hybridisation.
  • the DNA of the sample may now hybridize with the probes and, for this purpose, any known method capable of leading to a hybridisation stringency which is needed dependent on the probes may be employed.
  • interactions between the probes and the DNA of the liquid sample are determined. This may be done by using fluorescently labelled primers as set out above. Any other standard technique in the field may, however, also be used. Preferred is the use of a fluorescence-detecting device which can detect the fluorescence intensity over each hybridisation cycle employed.
  • the present invention also discloses a method of diagnosing an infectious disease in combination with the analysis of the genetic profile of the host by a) Providing a tissue sample of the host outside the human or animal body b) Detecting pathogens and host specific factors simultaneously in said tissue sample according to the method set out above relating to the detection of pathogen(s) and host specific factors c) Assigning the disease according to the pathogen(s) detected in step (b) d) Determining the susceptibility of the host to said disease and/or the prognosis of said disease e) Diagnosing the disease and predicting the host's reaction.
  • Said information may be obtained simultaneously according to the invention.
  • tissue sample may be comprised of blood of the host suspicious of an infection, as well as of other tissues where pathogens may be present. This comprises e.g. epithelial-derived tissues of the respiratory tract which is often the area of an infectious disease.
  • blood is used as sample of the host.
  • pathogens are present in the blood.
  • any method known to the skilled person in the art may be used. This may be for example the process of taking a blood sample of the host by using a sterile needle.
  • the method of detection as described in detail above may be carried out.
  • the disease may be determined by assigning a certain disease due to the presence of certain pathogens.
  • the susceptibility and/or prognosis of the host to said assigned disease may be determined according to the host-specific factors detected by the method described above according to the invention.
  • the information gained on the pathogen, the disease caused by said pathogen as well as on the susceptibility of the host to said disease and/or prognosis for the disease in the host/patient may be used for further decisions regarding any necessary medical steps.
  • a microarray device as well as the methods according to the invention may lead to very important information for clinical personnel on e.g. a patient suffering from a severe sepsis for a suited treatment as well as further observation steps which might be necessary.
  • the present invention may thus be used in a preferred embodiment in a clinical setup such as an emergency care unit in order to rapidly obtain information on pathogen(s) and genetic predispositions of the patient.
  • pathogens and the infectious diseases caused by said pathogens represent a major problem, such as the hospital-acquired sepsis.
  • a blood sample of a patient suspicious of an infection is obtained.
  • Total DNA is isolated from said sample.
  • a multistep PCR with fluorescently labelled primers is carried out to amplify regions of interest.
  • Said primers are designed according to the probes of the microarray and comprise primers specific for pathogen-DNA regions as well as for host-DNA regions of interest.
  • a region of interest may be the region surrounding position -308 of the human TNF ⁇ -gene (see Fig. 1).
  • the two primers depicted in Fig. 1 were used in the PCR-reaction (labelled with Cy5).
  • the amplified regions of interest are comprised in a liquid sample.
  • Said sample is now loaded into the chamber of a microarray device to allow for hybridisation between the amplified regions and the probes of the microarray device.
  • Said probes are printed on specific spots of the microarray.
  • probes 1-8 (as well as two control-probes 9 and 10) as listed in Fig. 1 and the table of Fig. 2 A were used.
  • Probes 1, 3, 5 and 7 have a G at the position corresponding to the SNP at position -308 of the human TNF ⁇ -gene and, therefore, represent the wildtype situation.
  • the probes were printed on the microarray device as schematically shown in Fig. 2 A and described in the legend of Fig. 2 and the sample was loaded into said microarray device.
  • Fig. 2 B shows the image of the fluorescence signals after 5 cycles of hybridisation of the microarray schematically depicted in Fig. 2 A to analyze the SNP at position -308 of the human TNF ⁇ -gene.
  • Probes 9 and 10 show the orientation of the microarray. In all four different setups of probe-arrangement, fluorescence signals were only detected for probes 1, 3, 5, and 7. Therefore, the amplified DNA contained regions with a G at position -308 of the human TNF ⁇ -gene. Therefore, the patient is homozygous wildtype for the corresponding position.
  • Fig. 3 depicts the hybridisation profile of three different patient-samples regarding their analysis of the SNP at position -308 of the human TNF ⁇ -gene.
  • the corrected intensities are shown for each hybridisation cycle.
  • the probes used were identical to probes 1-8 as described above.
  • Fig. 3 A only signals of the positions of probes 1, 3, 5 and 7 are detected. Therefore, the patient is homozygous wildtype for position -308 of the human TNF ⁇ -gene (the image of the microarray shown in Fig.2 B would thus correspond to the signals and intensities, respectively, detected after hybridisation cycle 5).
  • Fig. 3 B shows a different profile of the signals.
  • the patient is homozygous for the SNP at position -308 of the human TNF ⁇ -gene.
  • the profile shown in Fig. 3 C corresponds to a case, wherein the patient is heterozygous at position -308 of the human TNF ⁇ -gene. Signals of all probes except the blanc are detected and, therefore, amplified DNA carrying a G as well as DNA carrying an A at position -308 must be present.
  • the experimental data thus shows that the present invention can be used to analyze the presence/ absence of a SNP on the basis of hybridisation signals in the overall setup of a microarray detecting pathogen-DNA and host-DNA.

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Abstract

La présente invention concerne un procédé de détection simultanée de pathogènes et de profilage génétique de l'hôte. La présente invention concerne ainsi un dispositif à micropuces comprenant des oligonucléotides sondes destiné à détecter simultanément à la fois l'ADN du pathogène et l'ADN de l'hôte, sur une matrice unique. L'invention concerne en outre un procédé de détection simultanée de l'ADN du pathogène et de l'ADN de l'hôte comprenant une étape de préparation d'un échantillon liquide comprenant lesdits ADN, suivie par une étape de chargement de l'échantillon à l'intérieur de la chambre du dispositif à micropuces, afin de le mettre en contact avec les oligonucléotides sondes, cette étape étant suivie par une étape finale de détection des interactions entre les ADN et lesdits oligonucléotides sondes. La présente invention concerne également un procédé de diagnostic d'une maladie infectieuse et d'analyse de manière simultanée du profil génétique de l'hôte pour prédire notamment la réaction de l'hôte à ladite maladie et pour appliquer par exemple un régime personnalisé et/ou pour classifier le groupe de risque de l'hôte.
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CN102080127A (zh) * 2010-09-06 2011-06-01 广州阳普医疗科技股份有限公司 一种检测15种临床常见病原微生物的基因芯片
CN102507934A (zh) * 2011-11-02 2012-06-20 集美大学 产酸克雷伯氏菌的时间分辨荧光免疫检测法
CN102507934B (zh) * 2011-11-02 2014-02-26 集美大学 产酸克雷伯氏菌的时间分辨荧光免疫检测法
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WO2019013613A2 (fr) 2017-07-09 2019-01-17 Hakken Enterprise Sa De Cv Procédés et trousses permettant de déterminer un risque de cancer
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CN111235290A (zh) * 2020-03-27 2020-06-05 东莞市厚街医院 一种厌氧菌液相芯片检测方法
CN111235290B (zh) * 2020-03-27 2023-10-31 东莞市厚街医院 一种厌氧菌液相芯片检测方法
CN117660656A (zh) * 2024-02-01 2024-03-08 浙江省肿瘤医院 与胃癌预后相关的菌群标志物及其应用
CN117660656B (zh) * 2024-02-01 2024-05-10 浙江省肿瘤医院 与胃癌预后相关的菌群标志物及其应用

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