EP1198597A1 - Procede permettant de detecter des organismes de fa on specifique a l'espece - Google Patents

Procede permettant de detecter des organismes de fa on specifique a l'espece

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Publication number
EP1198597A1
EP1198597A1 EP99939394A EP99939394A EP1198597A1 EP 1198597 A1 EP1198597 A1 EP 1198597A1 EP 99939394 A EP99939394 A EP 99939394A EP 99939394 A EP99939394 A EP 99939394A EP 1198597 A1 EP1198597 A1 EP 1198597A1
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EP
European Patent Office
Prior art keywords
eukaryotes
prokaryotes
kit
primers
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99939394A
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German (de)
English (en)
Inventor
Guido Krupp
Peter Scheinert
Rainer SÖLLER
Ulrich Spengler
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Artus Gesellschaft fur Molekularbiologische Diagnostik und Entwicklung Mbh
Original Assignee
Artus Gesellschaft fur Molekularbiologische Diagnostik und Entwicklung Mbh
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Publication of EP1198597A1 publication Critical patent/EP1198597A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to methods for the species-specific detection of prokaryotes and eukaryotes and to kits for carrying out these methods.
  • the invention relates to a method for the species-specific detection of sepsis pathogens.
  • Prokaryotes - ribosomal DNA includes sequences for:
  • Eukaryotes - ribosomal DNA includes sequences for:
  • the lengths of the rRNA spacers are extremely variable and within a well-defined group of microorganisms, e.g. Mycoplasmas, length alone is a suitable means of classifying microorganisms in this group (e.g. mycoplasmas) and can also be used in part to identify individual species within this group.
  • a secondary length differentiation is generally necessary after a defined restriction enzyme treatment of the amplified spacers. After gel electrophoretic separation and hybridization with species-specific oligonucleotide probes, the individual species within a group of microorganisms result in a characteristic band pattern.
  • the object of the present invention is therefore to provide a method for the detection of organisms (prokaryotes and eukaryotes), e.g. in selected biological samples, which is easy to use and suitable for routine use in medical laboratories.
  • the method should also have a high degree of specificity in relation to a very large number of excitation groups, it being possible to distinguish between individual species within these groups in a simple, experimentally uncomplicated and inexpensive manner.
  • the invention thus relates to a method for the species-specific detection of prokaryotes or eukaryotes with the aid of nucleic acid amplification techniques, in which, first of all, the prokaryotes or eukaryotes to be detected are enriched, concentrated and / or increased in a biological sample / or the DNA present in the biological sample is isolated and / or enriched.
  • the DNA is amplified using a nucleic acid amplification technique (NAT), in which a region of the DNA is amplified using amplification primers which contain the sequences conserved for the organisms in question. fected, which is flanked by the conserved sequences.
  • NAT nucleic acid amplification technique
  • NAT nucleic acid amplification techniques
  • a sequencing primer is added to the (isolated) amplificates, which hybridizes with a region within the amplified sequences which is conserved in the prokaryotes or eukaryotes to be detected.
  • the sequencing primer of this process stage referred to as "mini-sequencing" is chosen so that different elongates are obtained for the prokaryotes or eukaryotes to be detected if chain termination polymerization is carried out in which 1, 2 or 3 of the four possible deoxyribonucleoside triphosphates are carried out (dNTPs) or one of the four possible dNTPs is replaced by a chain termination deoxyribonucleoside triphosphate (chain termination NTP).
  • dNTPs deoxyribonucleoside triphosphates
  • chain termination NTP chain termination deoxyribonucleoside triphosphate
  • one or more further sequencing primers are then used which hybridize / hybridize with a region / regions within the amplified sequences which are / are conserved in the prokaryotes or eukaryotes to be detected, the further / further sequencing primer (s) being selected such that different elongates are obtained for the prokaryotes or eukaryotes to be detected if chain termination polymerization is carried out using 1, 2 or 3 of the four possible dNTPs or of the four possible dNTPS one replaced by a chain termination NTP.
  • the sequencing primers used in chain termination polymerization are preferably 15 to 30 nucleotides in length.
  • chain termination NTPs chain terminators
  • chain terminators come e.g. Dideoxy-ribonucleoside triphosphates (ddNTPs), 3 'O-methyl-NTPs, 3' -A ino-NTPs and the like in question.
  • the method can be carried out using several sequencing primers by replacing one of the four possible dNTPs with a chain terminating NTP (for example ddA) in the chain termination reaction, the polymerization being carried out in the simultaneous presence All sequencing primers are carried out, the sequencing primers each bearing different markings in order to make it possible to differentiate the elongates.
  • ddA chain terminating NTP
  • the length of the elongates can be determined by methods known to those skilled in the art, e.g. using electrophoretic methods, by mass spectrometric detection or e.g. by fluorescence correlation spectroscopy (FCS) or comparable methods.
  • FCS fluorescence correlation spectroscopy
  • Suitable markings e.g. Fluorescence labels or labels differing in their mass are well known to the person skilled in the art.
  • the primer elongates are detected by mass spectroscopy, using MALDI-TOF spectrometry (Matrix-assisted laser desorption ipnization time of flight spectrometry (MALDI-TOF spectrometry), cf. for example Fu et al., Nature Biotechnol .16 (1998) 381-384)) has proven to be particularly advantageous, which allows detection of up to 2000 nucleotides or more.
  • MALDI-TOF spectrometry Microx-assisted laser desorption ipnization time of flight spectrometry
  • multiplex analysis is advantageous, ie the simultaneous analysis of the elongation products of several sequencing primers with different target sequences.
  • the elongation reactions with differently labeled sequencing primers are carried out either in one reaction vessel using a single terminator (alternative A) or in different reaction vessels using different terminators (alternative B), alternative B combining the products of the (parallel) elongation reactions for the analysis become.
  • the necessary differentiation of the elongation products is possible by marking the oligonucleotides dress with:
  • the method according to the invention is not subject to any methodological restrictions on a specific group of organisms.
  • the only prerequisite for reliable (re) recognition and organism differentiation is that the target sequences (e.g. rRNA, rDNA) of the organisms to be detected are known.
  • the inventive method of organism determination based on universal, i.e. Sequences that are essential in all organisms (such as ribosomal sequences) are therefore suitable for the differential detection of defined groups of organisms of all types of prokaryotes and eukaryotes (bacteria, fungi, plants and animals).
  • prokaryotes Representatives of the group of Bacteria and Archeae understood and included all species that fall under these groups.
  • eukaryotes includes both unicellular and multicellular organisms, such as, for example, amoebas, trypanosomes, plasmodia, yeasts, single and multicellular parasites, and also plants and animals, all species falling under these groups being included.
  • variable sequences flanked by conserved regions that are essential in all (to be detected) organisms can be considered as target sequences in the context of the present invention.
  • variable rDNA sequences including rRNA spacers as target sequences.
  • other species-specific genes or gene segments can also be assumed, e.g. of the cytochrome b gene from mitochondria (cf. Irwin et al., J. Mol. Evol. 32 (1991) 128-144), which can serve for the species-specific detection of eukaryotes.
  • the method according to the invention can be used in a wide variety of areas.
  • Fisheries e.g. spot checks when checking fishing quotas
  • food controls e.g. spot checks when checking fishing quotas
  • the method of the present invention can be used universally, e.g. to clarify the question of which pathogens (groups) are present when a specific disease symptom is observed.
  • the causes of the following medically relevant disease symptoms can be determined using the method according to the invention:
  • Fever as a result of bacterial (sepsis), parasitic (eg malaria) or fungal pathogens (eg candida) in the blood; Inflammation of the brain or meninges, ie headache, stiff neck, loss of consciousness due to bacterial (meningococcal, Haemophilus influenza, pneumococcal, tubercle bacilli, E. coli, Listeria monocytogenes), parasitic (e.g. toxoplasmosis) or fungal infections (e.g. cryptocansoc) infections c ;
  • Respiratory tract infections i.e. Cough, expectoration, shortness of breath etc. due to bacterial (e.g. pneumococcal, chlamydia, mycoplasma), parasitic or fungal infections (e.g. Pneumocystis carinii);
  • bacterial e.g. pneumococcal, chlamydia, mycoplasma
  • parasitic or fungal infections e.g. Pneumocystis carinii
  • Infections of the eye i.e. watery eye, possibly pus, cloudy vision, etc.
  • a number of bacterial e.g. chlamydia, gonorrhea, Staphylococcus aureus
  • parasitic pathogens e.g. Toxoplasma gondii, Onchocerca volvulus etc.
  • bacterial e.g. Salmonella, Yersinia, Campylobacter, E.coli, vibrions, clostridia, Bacillus
  • parasitic e.g. amoeba, Giardia, Cryptosporidia
  • fungal infections Candida
  • Pain during micturition, hematuria etc. as a sign of a urinary tract infection e.g. caused by E. coli, staphylococci, other Enterobacteriaces such as Proteus mirabilis as bacterial pathogen, Candida as fungus representative, or schistosomes as parasitic pathogen.
  • This group also includes primarily sexually transmitted agents, e.g. Chlamydia, gonorrhea, syphilis, mycoplasmas etc .;
  • Skin infections ie reddening of the skin, itching of the skin, blistering etc. attributable to fungal infections with dermatophytes such as Trichophyton, Epidermophyton and Microsporon, bacterial infections (eg Staphylococcus aureus or Steptococcus pyogenes) or parasitic diseases, e.g. Leishmania.
  • dermatophytes such as Trichophyton, Epidermophyton and Microsporon
  • bacterial infections eg Staphylococcus aureus or Steptococcus pyogenes
  • parasitic diseases e.g. Leishmania.
  • pathogens can be detected in a species-specific manner, as a result of which more targeted and thus often fewer side effects treatments are made possible than in the case of broader therapies.
  • biological samples are understood to mean all types of samples in which a limited, well-defined group of procarotes or eukaryotes to be detected can be present.
  • the biological sample may e.g. blood, stool, smears etc. (e.g. detection of sepsis pathogens in blood, detection of protozoa in blood or faeces, etc.).
  • animal or plant tissues or liquids such as e.g. Meat or meat juice, serve as a sample (e.g. detection of Salmonella in meat, detection of plant pests in seeds).
  • Environmental samples such as Soil or water samples into consideration.
  • fish stock analysis or e.g. fish spawning or a plankton sample can also serve as a biological sample for specifying species in fish farming and fishing biology.
  • a method for the detection of sepsis pathogens is thus provided, in which the biological sample is blood and the organisms from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Neisseria meningitidis, Escherichia coli, Enterobacter spec, Proteus spec, Pseudomon
  • the method is preferably carried out by taking blood from the patient in buffer and subjecting the human cells to lysis, for example using 1 to 20% by weight of Tween (polyoxyethylene derivatives of sorbitan esters), Triton or 3- [N- ( 3-Cholanamido-propyl) -dimethylammonio] -l-propanesulfonate (CHAPS).
  • Tween polyoxyethylene derivatives of sorbitan esters
  • the blood is particularly preferably taken up in lysis buffer by mixing 1 volume of blood with 4 volumes of lysis buffer, the lysis buffer consisting of 109.5 g (0.32 M) sucrose, 1.221 g (10 mM) Tris-HCl, 10 ml (1% ) Triton X-100, 1.016 g (5 mM) MgCl 2 ad 1000 ml distilled water (pH 7.5), then incubated for 5 minutes at room temperature and then the mixture in a centrifuge tube with a suitable amount of a cushion liquid of a density of Layer 1.07 g / ml and centrifuge at 1500 g for 30 minutes at room temperature, whereby the bacteria in the pellet are obtained (enriched).
  • the lysis buffer consisting of 109.5 g (0.32 M) sucrose, 1.221 g (10 mM) Tris-HCl, 10 ml (1% ) Triton X-100, 1.016 g (5 mM) MgCl 2 ad 1000
  • a pillow liquid is used, of which 100 ml from 10 ml (1.5 M) NaCl solution (87.6 g NaCl per 1000 ml), 49.2 ml Percoll with a density of 1.13 ⁇ 0.005 g / ml and 40.8 ml distilled water.
  • the pellet is then preferably washed twice with 0.15 M NaCl solution at room temperature, pelleted at 1500 g, and the bacterial pellet resuspended in TE buffer (10 mM Tris-HCl (pH 8), 1 mM Na 2 EDTA) is digested subsequently for 2 hours at 56 ° C. with Proteinase K, and the Proteinase K is inactivated then at 95 ° C. for 15 minutes, the lysate obtained, which contains the bacterial DNA, either being used directly in a nucleic acid amplification technique, or prior to further purification using, for example, a purification system based on glass matrix (glass milk) Digestion of bacteria and / or to eliminate inhibitors of amplification.
  • TE buffer 10 mM Tris-HCl (pH 8), 1 mM Na 2 EDTA
  • Sucrose or Ficoll can also be used instead of Percoll for the pillow fluid.
  • the density of the pillow should in any case be 1.07 g / ml.
  • the primer pair according to SEQ ID NO 1 and 2 or the primer pair according to SEQ ID NO 3 and 4 is used as the amplification primer when detecting sepsis pathogens, the primer pair according to SEQ ID NO 1 and 2 being particularly preferred.
  • the primer pair according to SEQ ID NO 1 and 2 being particularly preferred.
  • Three chain termination polymerizations are particularly preferably carried out, a reaction in the presence of the sequencing primers according to SEQ ID NO 5 and 6 (preferably with ddA as terminator), a reaction with the primers according to SEQ ID NO 7, 8 and 9 (preferably with ddG as terminator), and a reaction in the presence of the primers according to SEQ ID NO 10 and 11 (preferably with ddA as terminator).
  • NAT e.g. PCR
  • chain termination polymerization of the method according to the invention as for a conventional method with PCR and subsequent enzyme treatment (see e.g. Gürtler, cited above).
  • the detection or length determination of the elongates can be automated (e.g. by using MALDI-TOF spectrometry), the final analysis can be carried out in the seconds to minutes range.
  • a conventional gel analysis is naturally difficult to automate and therefore more expensive than the solution provided according to the invention.
  • the particular advantage of the method according to the invention is that, surprisingly, a simple and generally applicable method is made available, in which a species-specific detection of organisms is possible which - depending on the type of (biological) sample - a limited, well-defined group of Prokaryotes or eukaryotes.
  • the method for isolating and / or enriching bacterial DNA (including the preparation or concentration of bacteria) developed according to a partial aspect of the present invention can also be used as an isolated enrichment method in other areas of application - in particular of biological samples from the group consisting of blood or blood products , Meat juice, milk, (waste) water or any other liquid that may be contaminated with bacteria.
  • the method is characterized in that the biological sample is optionally taken up in buffer and the non-bacterial cells are subjected to lysis, for example using 1 to 20% by weight of tween (polyoxyethylene derivatives of sorbitan esters), triton or 3- [N- (3-Cholanamidopropyl) dimethylammonio] 1-propanesulfonate (CHAPS).
  • tween polyoxyethylene derivatives of sorbitan esters
  • CHAPS 3- [N- (3-Cholanamidopropyl) dimethylammonio] 1-propanesulfonate
  • the preferred lysis buffer mentioned above can also be used in the same or similar volume fractions. After incubation for 5 minutes at room temperature, the mixture is then overlaid in a centrifuge tube with a suitable amount of a pillow liquid (see above).
  • the procedure is preferably as described above using the example of the sepsis pathogen.
  • the lysate obtained in this way can, for example, either be used directly in a nucleic acid amplification technique; if appropriate, a further purification can be carried out by using a purification system based, for example, on a glass matrix for disrupting bacteria and / or for eliminating amplification inhibitors.
  • the advantages of the enrichment process according to the invention are that the separation of a large, e.g. amount of DNA derived from blood cells is made possible. Furthermore, the removal of e.g. substances derived from blood (heme etc.) that can inhibit the feasibility of nucleic acid amplification techniques.
  • kits for carrying out the above-mentioned methods are also provided.
  • the lysis buffer of the kit particularly preferably consists of 109.5 g (0.32 M) sucrose, 1.221 g (10 mM) Tris-HCl, 10 ml (1%) Triton X-100, 1.016 g (5 mM) MgCl 2 ad 1000 ml of distilled water (pH 7.5).
  • the pillow fluid of the kit preferably consists of 10 ml 1.5 M NaCl solution (87.6 g NaCl per 1000 ml), 49.2 ml Percoll with a density of 1.13 + 0.005 g / ml, 40.8 ml distilled Water (100 ml pillow liquid).
  • kits for carrying out the method for isolating and / or enriching bacterial DNA can optionally contain further constituents which are required or are expedient for (further) sample preparation.
  • the kits additionally contain components for disrupting the bacteria and, if appropriate, for DNA purification (in particular for eliminating amplification inhibitors) or the like, e.g. Components of glass matrix-based commercial purification systems (e.g. QIAamp DNA Kit).
  • Such a kit preferably also contains proteinase K for disrupting the bacteria.
  • a kit for performing the method for species-specific detection of prokaryotes or eukaryotes from biological or environmental samples contains - if necessary in addition to Components of an above-mentioned kit for carrying out the DNA processing or enrichment process - the following components:
  • sequencing primers preferably with a length of 15 to 30 nucleotides
  • the kit contains either 1, 2 or 3 of the four possible deoxyribonucleoside triphosphates (dNTPs) or all four possible dNTPs, a dNTP being replaced by a chain termination deoxyribonucleoside triphosphate (such as, for example, a ddNTP, 3'O-methyl -NTP, 3 '-amino-NTP or the like) is replaced.
  • dNTPs deoxyribonucleoside triphosphates
  • a chain termination deoxyribonucleoside triphosphate such as, for example, a ddNTP, 3'O-methyl -NTP, 3 '-amino-NTP or the like
  • the kit optionally also includes components for determining the elongate length (for example for performing electrophoresis) or corresponding components which are necessary or useful in order to prepare the samples for corresponding methods with which the elongate length can be determined.
  • components for determining the elongate length for example for performing electrophoresis
  • corresponding components which are necessary or useful in order to prepare the samples for corresponding methods with which the elongate length can be determined.
  • the kit is, for example, a kit for the detection of prokaryotes, in particular of sepsis pathogens, which as amplification primer contains two highly conserved sequences from the rRNA region, preferably the amplification primers according to SEQ ID NO 1 and 2 or according to SEQ ID NO 3 and 4 (the primer pair according to SEQ ID NO 1 and 2 being particularly preferred), and / or at least one sequencing primer which hybridizes with a conserved region of the rRNA region, preferably at least one sequencing primer from the group consisting of SEQ ID NO 5, 6, 7, 8, 9, 10 and 11 (mixtures or mixtures of the sequencing primers according to SEQ ID NO 5 and 6, 7 to 9 and 10 and 11 in a single or separate container are particularly preferred ) contains.
  • amplification primer contains two highly conserved sequences from the rRNA region, preferably the amplification primers according to SEQ ID NO 1 and 2 or according to SEQ ID NO 3 and 4 (the primer pair according to SEQ ID NO 1 and 2 being particularly preferred), and /
  • bacteria from blood were prepared or enriched.
  • blood was first taken up in lysis buffer, which consisted of 109.5 g (0.32 M) sucrose, 1.221 g (10 mM) Tris-HCl, 10 ml (1%) Triton X-100, 1.016 g (5 M) MgCl 2 to 1000 ml of distilled water (pH 7.5) consisted of one part of blood mixed with four parts of lysis buffer (eg 3 ml of blood with 12 ml of lysis buffer) and incubated for 5 minutes at room temperature.
  • the blood-lysis buffer mixture was then placed on a cushion (5 ml) and the entire solution at 1500 g for 30 minutes centrifuged at room temperature (RT).
  • 100 ml of the pillow liquid is composed as follows: 10 ml (1.5 M) NaCl solution (87.6 g NaCl per 1000 ml), 49.2 ml Percoll with a density of 1.13 g / ml (+ / - 0.005 g / ml), 40.8 ml distilled water. Sucrose or Ficoll can also be used instead of Percoll.
  • the density of the pillow should in any case be 1.07 g / ml.
  • the pellet in which the bacteria were found was washed twice at RT with 0.15 M NaCl and pelleted at 1500 g.
  • proteinase K digestion of the bacterial pellet resuspended in TE buffer (10 M Tris-HCl (pH 8), 1 mM Na 2 EDTA) took place at 56 ° C. for two hours, and the proteinase K was subsequently at 95 ° C. for Disabled for 15 minutes.
  • the lysate obtained was then used in the amplification.
  • commercial purification systems based on glass matrix eg QIAamp DNA Kit
  • Example 1 The bacterial DNA obtained in Example 1 was subjected to PCR amplification, and the amplificates obtained were then used in a chain termination polymerization.
  • PCR amplification The bacterial DNA obtained in Example 1 was subjected to PCR amplification, and the amplificates obtained were then used in a chain termination polymerization.
  • These methods and the conditions under which these methods are carried out are well known to the person skilled in the art (cf., for example, Garcia-Pichel et al., Arch. Microbiol. 169 (1998) 469-482; Cha et al., PCR Methods and Application 3 (1993) S18-29 (Manual Supplement); Tracy et al., Bio Techniques 11 (1) (1991) 68-75).
  • A. Nucleic acid amplification A. Nucleic acid amplification:
  • the 16S rRNA region was amplified with the aid of PCR using two highly conserved primers.
  • a 16S-5 'terminal (SEQ ID NO 1) and a 16S-3' terminal primer (SEQ ID NO 2) were combined, one of the two PCR primers (here the 16S- 5 'terminal primer) being used for immobilization suitable derivative, e.g. Biotin contained.
  • the 16S-23S rRNA spacer can also be amplified, optionally with the primers according to SEQ ID NO 3 (16S proximal primer) and SEQ ID NO 4 (23S proximal primer).
  • the aplified rRNA areas were subsequently subjected to an elongation.
  • oligonucleotides were used as sequencing primers, which are universally preserved or at least are present in all target organisms within the group to be detected. After elongation with a polymerase - leaving out one of the four natural nucleoside triphosphates or in the presence of a terminator nucleoside triphosphate (e.g. ddNTP) - products with different, characteristic lengths were created.
  • a polymerase e.g. ddNTP
  • sequencing primers which are complementary to 16S rRNA sequences which are universal in all bacteria (Bacteria and Archaea), were used for elongation in the present case (see also DJ Lane, loc. Cit.): 109r: SEQ ID NO 5 (109rl) and SEQ ID NO 6 (109r2) 685r: SEQ ID NO 7 (685rl), SEQ ID NO 8 (685r2), SEQ ID NO 9 (685r3).
  • 1475r s is - within the sepsis pathogen - specific for the group of gram-positive cocci. 1475r is - within the sepsis pathogen - specific for the group Haemophilus.
  • the corresponding 16S rRNA sequences were selected because there are very divergent sequences directly adjacent, i.e. Sequences that can be used for a species-specific differentiation of microorganisms (bacteria).
  • nucleoside triphosphates dGTP, dCTP, dTTP were used either alone or with the addition of ddATP (as indicated in Table 1).
  • dGTP, dCTP and dTTP alone, the values given in Table 1 must be reduced by one nucleotide unit.
  • nucleoside triphosphates dATP, dCTP, dTTP were used either alone or with the addition of ddGTP (as indicated in Table 1).
  • dGTP, dCTP and dTTP alone, the values given in Table 1 must be reduced by one nucleotide unit.
  • nucleoside triphosphates dGTP, dCTP, dTTP were used either alone or with the addition of ddATP (as indicated in Table 1).
  • dGTP, dCTP and dTTP alone, the values given in Table 1 must be reduced by one nucleotide unit.
  • the oligonucleotide elongates obtained with the above-mentioned primers are shown in Table 1 below.
  • the elongates were detected by fluorescence detection or mass spectrometry.
  • Primer 1475r can be used (see Table 1).

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Abstract

La présente invention concerne un procédé permettant de détecter, de façon spécifique à l'espèce, des procaryotes et des eucaryotes, ainsi que des trousses permettant de mettre en oeuvre ce procédé. Cette invention concerne notamment un procédé permettant de détecter, de façon spécifique à l'espèce, des agents inducteurs de sepsie.
EP99939394A 1999-07-22 1999-07-22 Procede permettant de detecter des organismes de fa on specifique a l'espece Withdrawn EP1198597A1 (fr)

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PCT/EP1999/005234 WO2001007648A1 (fr) 1999-07-22 1999-07-22 Procede permettant de detecter des organismes de façon specifique a l'espece

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