WO2001007648A1

WO2001007648A1 - Method for the species-specific detection of organisms

Info

Publication number: WO2001007648A1
Application number: PCT/EP1999/005234
Authority: WO
Inventors: Guido Krupp; Peter Scheinert; Rainer SÖLLER; Ulrich Spengler
Original assignee: Artus Gesellschaft für molekularbiologische Diagnostik und Entwicklung mbH
Priority date: 1999-07-22
Filing date: 1999-07-22
Publication date: 2001-02-01
Also published as: EP1198597A1; AU5371099A

Abstract

The invention relates to a method for the species-specific detection of prokaryotes and eucaryotes, and to kits for implementing said method. The invention also relates to a method for species-specific detection of sepsis inducers.

Description

Procedure for the species-specific detection of organisms

The present invention relates to methods for the species-specific detection of prokaryotes and eukaryotes and to kits for carrying out these methods. In particular, the invention relates to a method for the species-specific detection of sepsis pathogens.

The reliable detection of a wide variety of organisms, especially microorganisms such as bacteria, plays an increasing role in medical microbiology and is often a prerequisite for the targeted treatment of infections in humans and animals. In this context, Gürtler et al. (Microbiology 142 (1996) 3-16) and Scheinert et al. , J. Microbiol. Method 26 (1996) 103-117) proposed the use of the 16S-23S rDNA or rRNA spacer region for the typing and identification of bacteria. The sequences of rRNAs have so far been used most frequently for the identification of microorganisms. In the genome of prokaryotic and eukaryotic organisms, the operons for riboso ale RNAs (rRNAs) are organized in such a way that

BESTATIGUNGSKOPIE First, a comprehensive precursor rRNA is transcribed. These precursor rRNAs essentially contain the following components in the 5 '-3' direction:

Prokaryotes - ribosomal DNA includes sequences for:

5 '- 16S rRNA - transcribed 16S / 23S rRNA spacer - 23S rRNA - transcribed 23S / 5S rRNA spacer - 5S rRNA - 3'

Eukaryotes - ribosomal DNA includes sequences for:

5 '- 18S rRNA - transcribed 18S / 5.8S rRNA spacer (ITS1) - 5.8S rRNA - transcribed 5.8S / 28S rRNA spacer (ITS2) - 28S rRNA - 3'.

This general organization is present in all prokaryotes (Bacterla and Archaea) and eukaryotes (see e.g. B. Lewin, Genes V, Cell Press, Cambridge, Massachusetts / USA, 1994), with very few exceptions so far, e.g. the Archaeon Thermoplasma acldophllum (Achenbach-Richter et al., System. Appl. Microbiol. 10 (1988) 211-214) or Helicobacter (Tomb et al., Nature 388 (1997) 539-547) are known.

It has been found that 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.

In the case of Gürtler et al. (loc. cit.) and Scheinert et al. (op. cit.) described method for typing and identification of bacteria using the 16S-23S rDNA or rRNA spacer region is first an application by means of suitable primers by polymerase chain reaction (PCR Amplification). The amplified spacers can then be separated by gel electrophoresis and detected by suitable detection methods, such as, for example, staining with ethidium bromide or silver, or by fluorescence detection. The position of the bands, ie the length of the amplified spacers, allows a preliminary conclusion to be drawn about the respectively detected microorganism or the respective group of microorganisms.

For reliable detection, in particular for the detection of individual species, 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.

These methods have the disadvantage that a precise knowledge of the band pattern to be expected in each case is required for the species-specific detection of pathogens and, moreover, an extremely large number of specific oligonucleotide probes must be available. The methods known to date in the prior art are therefore not suitable for routine detection of pathogens in medical laboratories.

With regard to sepsis, a severe, acute infectious disease caused by certain bacteria that have entered the bloodstream, there is also the additional problem that these bacteria originate from a locally inflammatory focus and via the blood or lymphatic system be scattered in batches. Due to this cyclical fluctuation, relatively large amounts of blood are required for bacterial detection in sepsis (approx. 5 to 10 ml each for aerobic and anaerobic blood culture). This large sample volume can only be handled with great effort using the currently available means and methods, since larger contaminations with foreign DNA (ie non-bacterial DNA) lead to unspecific detection. purification and thus leads to a reduction in the specificity of a subsequent PCR. The large number of red blood cells can also have an inhibitory effect on PCR, and larger sample volumes are generally not suitable for commercially available kits, since the columns used for purification are often clogged.

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. Finally, it is an object of the invention to provide a simple method which enables the detection of microorganisms which are only present in low concentrations, as is e.g. is occasionally the case in the cyclical course of sepsis.

The object is achieved according to the invention by methods according to claims 1 to 17 and 19 to 24 and kits according to claims 25 to 40.

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.

Any nucleic acid amplification techniques (NAT) known to those skilled in the art can be used to carry out the method according to the invention, preferably a NAT from the group consisting of PCR, bDNA, LCR, 3SR, SDA or NASBA.

In the next step, 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). After determining the length increase obtained in each case (elongates, ie the number of bases by which the amplified products have been extended), which the products of chain termination polymerization have compared to the amplified products, these values are compared with the elongates which are relevant for the respective organisms due to their sequence are to be expected. A certain prokaryote or eukaryote present in the sample is detected by the fact that the elongation observed (achieved) and the expected elongation match.

In individual cases, it can happen that several of the organisms present in the sample use the same sequencing primer to give the same elongates (ie the same increase in length compared to the amplificates). If appropriate, 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. After determining the length increase (elongates; number of bases by which the amplified products have been extended) for each chaining primer, which the products of chain termination polymerization have compared to the amplified products, these values are compared with the elongates, which for the respective organisms are due to their sequence are to be expected. A prokaryote or eukaryote present in the sample is detected by the fact that the elongation values (experimentally determined elongation pattern) obtained experimentally using the different sequencing primers match the elongation values to be expected for this organism (expected elongation pattern).

For the species-specific detection of organisms in biological samples, exactly as many different sequencing primers must be used until different elongation patterns are obtained for all organisms that may be present in the sample. This is illustrated in the example section using the determination of sepsis pathogens.

The sequencing primers used in chain termination polymerization are preferably 15 to 30 nucleotides in length. As chain termination NTPs (chain terminators) come e.g. Dideoxy-ribonucleoside triphosphates (ddNTPs), 3 'O-methyl-NTPs, 3' -A ino-NTPs and the like in question.

According to a particular embodiment of the invention, 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.

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. In this context, it is advantageous to mark the sequencing primer. When using several sequencing primers, these should be marked differently, especially when carrying out the polymerizations in a single reaction vessel using only one terminator (e.g. ddA). Suitable markings, e.g. Fluorescence labels or labels differing in their mass are well known to the person skilled in the art.

According to a particularly preferred embodiment of the invention, 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.

In this context, "multiplex analysis" is advantageous, ie the simultaneous analysis of the elongation products of several sequencing primers with different target sequences. For this purpose, 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:

(a) 5 'terminal additional sequences of different lengths, "Tails" (can be used in all detection methods),

(b) Different 5'-terminal additions which drastically change running behavior (in standard polyacrylamide gel electrophoresis or high-resolution gel electrophoresis such as plate gel, capillary or array capillary gel electrophoresis) or molecular weight (MALDI-TOF). Examples of such additions are polyethylene glycol chain, cholesterol derivatives, etc. .

(c) different fluorescent dyes (with standard polyacrylamide gel electrophoresis, combined with a fluorescence scanner; routinely with high-resolution gel electrophoresis; in principle also possible with mass spectroscopic analysis, whereby differing overlapping elongation patterns are differentiated by characteristic molecular weight values and the extremely high resolution can be).

In principle, 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).

In the context of the present invention under "prokaryotes" Representatives of the group of Bacteria and Archeae understood and included all species that fall under these groups. The term "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.

In principle, all 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. For example, it is possible to use the variable rDNA sequences including rRNA spacers as target sequences. In addition, 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. However, the (micro) organisms mentioned in the following application examples are only representative of other representatives from the respective organism kingdoms (prokaryota = bacteria; eukaryota = single and multiple fungi, plants and animals) as they are distinguished in the biological system:

a) Detection of sepsis pathogens and other bacterial pathogens in humans, animals and plants and in cell cultures thereof;

b) Detection of unicellular organisms (protozoa: flagellates, amoebas, sporozoa, ciliates) as the cause of e.g. Intestinal diseases in humans and animals (especially also farm animals);

c) Detection of pests, contaminants, spoilage agents and pathogens in agriculture, animal husbandry, the food industry and seeds: here again defined groups of organisms from all systematic groups of life (see above) are differentiable and expressly included, especially from the groups of bacteria, protozoa, fungi and artrophodes;

d) Evidence of helminths (worms) as the cause of intestinal diseases (e.g. diarrhea);

e) detection of fungi as pathogens of fungal diseases in humans, animals and plants;

f) determination of species, race and origin in animal and plant breeding, species conservation breeding and agriculture,

Fisheries (e.g. spot checks when checking fishing quotas) and food controls;

g) Biodiversity studies for defined groups of organisms such as of freshwater or saltwater fish including their larval stages in Europe or other geographically delimitable regions (e.g. to determine stocks for catch quotas etc.), determination of lead organisms for water quality or soil assessment.

In particular in medical diagnostics, 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. For example, 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);

Infections of the eye, i.e. watery eye, possibly pus, cloudy vision, etc. Again, a number of bacterial (e.g. chlamydia, gonorrhea, Staphylococcus aureus) or parasitic pathogens (e.g. Toxoplasma gondii, Onchocerca volvulus etc.) can be considered as possible causes;

- diarrhea and weight loss etc. as a result of bacterial (e.g. Salmonella, Yersinia, Campylobacter, E.coli, vibrions, clostridia, Bacillus), parasitic (e.g. amoeba, Giardia, Cryptosporidia) or 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.

By using the method according to the invention, 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.

In the context of the present invention, “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. When using the method according to the invention in medical diagnostics, 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.). In the detection of food contaminants, plant pests and the like, 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. For 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.

The person skilled in the art is able to select the biological samples for the species-specific detection of organisms in such a way that the group of organisms which may be present in the sample is limited and well-defined. For example, when a sepsis is being diagnosed, the pathogens responsible for this disease are known, so that the clinical suspicion of sepsis can be confirmed when examining a blood sample if one or more of the bacteria listed in Table 1 are detected by using the method according to the invention , According to a particular embodiment of the invention, 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, Pseudomonas ae ruginosa, Pseudomonas fluorescens, Pseudomonas mendocina, Pseudomonas syringae, Haemophilus influenzae, Haemophilus ducreyi and Bacteroides spec. are selected. 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). 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). According to a preferred embodiment of the invention, 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.

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.

Subsequently - as stated above - a NAT known to the person skilled in the art with subsequent chain termination polymerization is carried out.

According to a preferred embodiment of the invention, 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. For chain termination polymerization, preference is given to using at least one sequencing primer from the group consisting of SEQ ID NO 5, 6, 7, 8, 9, 10 and 11 (cf. also DJ Lane in "Nucleic Acid Techniques in Bacterial Systematics", John Wiley & Sons 1991, pp. 115-175). 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).

The advantages of the method of the present invention over the length determination of PCR fragments in an agarose gel with and without enzymatic restriction digestion are in particular to name increased specificity (certainty of detection). The use of an internal primer and its elongation (for mini-sequencing, see above) results in specific recognition of the PCR fragment and thus increased security in the sense of a clear statement compared to the mere length determination of PCR fragments without identity control, such as with Gürtler et al. (see Microbiology 142 (1996) 3-16; W096 / 19585).

It takes approximately the same time to carry out NAT (e.g. PCR) and 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). However, since 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. In contrast, a conventional gel analysis is naturally difficult to automate and therefore more expensive than the solution provided according to the invention.

Furthermore, 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. How already mentioned, 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). Alternatively, 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). In the further processing, including proteinase K digestion and subsequent proteinase K inactivation, 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.

Within the scope of the present invention, kits for carrying out the above-mentioned methods are also provided.

Contains a kit for carrying out the method for the preparation and / or concentration of bacteria from biological samples - in particular of blood or blood products, meat juice, milk, (waste) water or any other liquid which may be contaminated with bacteria a) lysis buffer containing 1 to 20% by weight of tween (polyoxyethylene derivatives of sorbitan esters), Triton® or 3-

[N- (3-cholanamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS),

b) centrifuge tubes,

c) Pillow fluid with a density of 1.07 g / ml, which is used to layer the biological sample over it.

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).

The 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:

a) Vessels and components for carrying out a NAT, including the amplification primers, the amplification primers containing sequences which are conserved in the relevant prokaryotes or eukaryotes to be detected,

b) Vessels and components for carrying out one or more chain termination polymerizations, including one or more (optionally labeled or differently labeled) sequencing primers (preferably with a length of 15 to 30 nucleotides) which hybridize with a region within the sequences to be amplified / hybridize, which is conserved in the prokaryotes or eukaryotes to be detected.

To carry out the chain termination polymerizations, 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.

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. Such constituents result for the person skilled in the art from the above description of the method for the species-specific detection of prokaryotes or eukaryotes, to which express reference is made in this connection. According to a particular embodiment of the invention, 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.

The present invention is explained in more detail below with the aid of examples.

example 1

Procedure for the enrichment of microorganisms in biological samples:

In the present example, bacteria from blood were prepared or enriched. For this purpose, 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.

Then 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. Alternatively, commercial purification systems based on glass matrix (eg QIAamp DNA Kit) can also be used to digest the bacteria and to eliminate inhibitors of the amplification.

Example 2

Detection of microorganisms by PCR-supported amplification and subsequent elongation of the amplificates:

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:

The 16S rRNA region was amplified with the aid of PCR using two highly conserved primers. For this purpose, 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.

Instead of the 16S rRNA, 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).

It is obvious to the person skilled in the art that the result of the PCR is not fundamentally influenced by modification of the oligonucleotides (wobbles, mismatches, biotin, digoxygenin or fluorescein labeling, etc.).

The aplified rRNA areas were subsequently subjected to an elongation.

B. Elongation of the amplificates

To elongate the amplified spacer sequences, 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.

The following 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).

Two differently labeled sequencing primers 1475r ^s (SEQ ID NO 10) and 1475r ^h (SEQ ID NO 11) were also used for elongation.

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).

For sequencing primer 109r, the nucleoside triphosphates dGTP, dCTP, dTTP were used either alone or with the addition of ddATP (as indicated in Table 1). When using dGTP, dCTP and dTTP alone, the values given in Table 1 must be reduced by one nucleotide unit.

For sequencing primer 685r, the nucleoside triphosphates dATP, dCTP, dTTP were used either alone or with the addition of ddGTP (as indicated in Table 1). When using dGTP, dCTP and dTTP alone, the values given in Table 1 must be reduced by one nucleotide unit.

For sequencing primer 1475r, the nucleoside triphosphates dGTP, dCTP, dTTP were used either alone or with the addition of ddATP (as indicated in Table 1). When using dGTP, dCTP and dTTP alone, the values given in Table 1 must be reduced by one nucleotide unit.

The elongation reactions were common among those skilled in the art known conditions carried out (see, for example, Fu et al., loc. cit.).

The oligonucleotide elongates obtained with the above-mentioned primers are shown in Table 1 below.

In the present example, the elongates were detected by fluorescence detection or mass spectrometry. A clear differentiation between Pseudomonas aeruginosa (elongates of 10 and 4 bases) and Haemophilus influenzae (elongates of 6 and 3 bases) was possible (see Table 1).

The data set initially obtained using primers 109r and 685r according to Example 2 made it impossible to distinguish between Staphylococcus aureus and Staphylococcus epidermidis. However, this is indispensable and was achieved through the additional use of the group-specific primer 1475r ^s . A distinction was also made between the two Haemophilus species

Primer 1475r can be used (see Table 1).

The results shown in Tab. 1 show that when using the sequence-specific primers, characteristic elongates are obtained which allow differentiation of different pathogens.

About 80% of all typical sepsis cases are recorded with the species listed in Table 1 (Geerdes-Fenge et al. (1994) Chemother. J. 3, 131-143). Detection of sepsis is therefore possible using only three sequence-specific primers, preferably 109r, 685r and 1475r. Tab. Sequence-specific oligonucleotide elongates within 16S rRNÄ regions

'"Mixture of 109rl and 109r2, ^b) Mixture of 685rl, 685r2 and 685r3, ^c) Mixture of 1475r ^s and 1475r ^h (14)": Only one isolate in this relatively heterogeneous genus gives this elongate length.

Claims

claims:

Method for the species-specific detection of prokaryotes or eukaryotes with the aid of nucleic acid amplification techniques, characterized in that the following steps are carried out:

a) amplification of the DNA from biological samples using a nucleic acid amplification technique,

amplifying a region of DNA delimited by conserved sequences using amplification primers containing the conserved sequences,

b) adding a sequencing primer to the amplificates which hybridizes with a region within the amplified sequences which is conserved in the prokaryotes or eukaryotes to be detected, the sequencing primer being chosen such that different elongates are obtained for the prokaryotes or eukaryotes to be detected if a chain termination polymerization is carried out using 1, 2 or 3 of the four possible dNTPs or one of the four possible dNTPs is replaced by a chain termination NTP,

c) determining the length of the elongates obtained,

the prokaryotes or eukaryotes being detected by the elongates obtained matching the elongates to be expected for the respective prokaryotes or eukaryotes based on their sequence,

d) one or more further sequencing primers optionally being used which hybridize with a region / regions within the amplified sequences sert / hybridize which is / are preserved in the prokaryotes or eukaryotes to be detected,

the further / further sequencing primer is / are selected such that different elongates are obtained in each case for the prokaryotes or eukaryotes to be detected, if a 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,

and wherein by using the further sequencing primer / sequencing primer different elongate patterns are obtained for the respective prokaryotes or eukaryotes,

e) determining the length of the elongates obtained,

the prokaryotes or eukaryotes being detected by the fact that the elongate patterns obtained when using several sequencing primers match the elongate patterns to be expected for the respective prokaryotes or eukaryotes based on their sequence.

2. The method according to claim 1, characterized in that one carries out an enrichment, concentration and / or multiplication of the prokaryotes or eukaryotes to be detected and / or isolation and / or enrichment of the DNA before the amplification.

3. The method according to claim 1 or 2, characterized in that nucleic acid amplification technology is selected from the group consisting of PCR, bDNA, LCR, 3SR, SDA or NASBA.

4. The method according to claims 1 to 3, characterized in that several sequencing primers are used and one of the four possible dNTPs is replaced by a chain terminating NTP in the chain termination reaction, the polymerization being carried out in the presence of all sequencing primers, the sequencing primers in each case wear different markings.

5. The method according to claims 1 to 4, characterized in that the sequencing primer has / have a length of 15 to 30 nucleotides.

6. The method according to claims 1 to 5, characterized in that the chain termination NTP is dideoxyribonucleoside triphosphate (ddNTP), 3 'O-methyl-NTP or 3' -amino-NTP.

7. The method according to claims 1 to 6, characterized in that the length of the elongates is determined by means of electrophoresis methods, by mass spectrometric detection or by fluorescence correlation spectroscopy.

8. The method according to claim 7, characterized in that the length of the elongates is determined by matrix-assisted laser desorption ionization time of flight spectrometry (MALDI-TOF spectrometry).

9. The method according to claims 1 to 8, characterized in that the amplified DNA region is rDNA.

10. The method according to claim 9, characterized in that the DNA region is the rRNA spacer region.

11. The method according to claims 1 to 9, characterized in that 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.

12. The method according to claims 1 to 11, characterized in that one carries out at least one chain termination polymerization and the / the sequencing primer from the group consisting of sequencing primers according to SEQ ID NO 5, 6, 7, 8, 9, 10 and 11 is selected.

13. The method according to claims 1 to 8, characterized in that it is a method for the species-specific detection of eukaryotes and the amplified DNA region is the cytochrome b gene from mitochondria.

14. The method according to claims 1 to 12, characterized in that it is a method for the detection of sepsis pathogens, 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, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mendocina, Pseudomonas syringae, Haemophilus influenzae, Haemophilus ducreyi and Bacteroides spec. are selected.

15. The method according to claim 14, characterized in that blood is taken up in a suitable amount of lysis buffer by mixing 1 volume part of blood with 4 volume parts 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, overlay a suitable amount of a pillow liquid, with 100 ml of the pillow liquid consisting 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 of distilled water, and centrifuged at 1500 g for 30 minutes at room temperature, the pellet was washed twice with 0.15 M NaCl solution is washed at room temperature, pelleted at 1500 g and the bacterial pellet resuspended in TE buffer is subsequently digested with Proteinase K for 2 hours at 56 ° C and then Proteinase K at 95 ° C for 15 Minutes inactivated, either using the lysate obtained directly in a nucleic acid amplification technique or carrying out further purification beforehand by using a glass matrix-based purification system to digest bacteria and / or to eliminate amplification inhibitors.

16. The method according to claims 1 to 10, characterized in that it is a method for the detection of protozoa, in which the biological sample is blood and the eukaryotes from the group consisting of the group consisting of flellates, amoebas, sporozoa and Ciliates are selected.

17. The method according to claims 1 to 11, characterized in that it is a method for the detection of Salmonella, in which the biological sample is meat or meat juice and the prokaryotes are Salmonella.

18. The method according to claims 1 to 10, characterized in that the biological sample is fish spawn and the eukaryotes are fish.

19. Use of a method according to claims 1 to 15 in medical diagnostics.

20. A process for the preparation and / or concentration of bacteria, characterized in that a biological sample is optionally taken up in buffer and existing non-bacterial cells using 1 to 20% by weight.

®% Tween (polyoxyethylene derivatives of sorbitan esters), Tritone ® or 3- [N- (3-cholanamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS) and incubation for 5 minutes Lysed at room temperature, then overlayed the mixture in a centrifuge tube with a suitable amount of a pillow liquid having a density of 1.07 g / ml and centrifuged at 1500 g for 30 minutes at room temperature, whereby the bacteria in the pellet were obtained and / or accumulated.

21. The method according to claim 20, characterized in that the pellet obtained is further washed twice with 0.15 M NaCl solution at room temperature and pelletized at 1500 g.

22. The method according to claim 20 or 21, characterized in that a cushion liquid is used, of which 100 ml from 10 ml (1.5 M) NaCl solution (87.6 g NaCl to 1000 ml), 49.2 ml Percoll with a density of 1.13 ± 0.005 g / ml and 40.8 ml of distilled water.

23. A method for isolating and / or enriching bacterial DNA, characterized in that one carries out a method according to one of claims 20 to 22 and the bacteria in the pellet are then subjected to lysis.

24. The method according to claim 23, characterized in that one carries out the lysis by resuspending the bacterial pellet in TE buffer and digesting with proteinase K for 2 hours at 56 ° C and then the proteinase K at 95 ° C for 15 Minutes inactivated to obtain a lysate containing the bacterial DNA.

25. A method for the species-specific detection of prokaryotes or eukaryotes with the aid of nucleic acid amplification techniques, characterized in that first a method according to claims 20 to 24 and then a method according to claims 1 to 14 is carried out.

26. Kit for performing a method according to claims 20 to 22, characterized in that it

a) Lysis buffer containing 1 to 20 wt .-% Tween ® (polyoxyethylene derivatives of sorbitan esters), Triton ® or 3- [N-

(3-Cholanamidopropyl) dimethylammonio] -1-propanesulfonate

(CHAPS),

b) centrifuge tubes, and

c) pillow fluid with a density of 1.07 g / ml

contains.

27. Kit according to claim 26, characterized in that the lysis buffer from 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).

28. Kit according to claim 20 or 27, characterized in that the pillow liquid from 10 ml (1.5 M) NaCl solution (87.6 g NaCl to 1000 ml), 49.2 ml Percoll with a density of 1.13 + 0.005 g / ml, 40.8 ml distilled water (per 100 ml pillow liquid).

29. Kits for carrying out a method according to claim 23 or 24, characterized in that it contains, in addition to the components of a kit according to claims 26 to 28, components for disrupting the bacteria and for DNA purification.

30. Kit according to claim 29, characterized in that it contains Proteinase K.

31. Kit for performing a method according to claims 1 to 18, characterized in that it

b) Vessels and components for carrying out one or more chain termination polymerizations, including one or more (optionally labeled or differently labeled) sequencing primers (preferably with a length of 15 to 30 nucleotides) which hybridize with a region within the sequences to be amplified / - hybridize which is conserved in the prokaryotes or eukaryotes to be detected, and

c) Components for determining the elongate length

contains.

32. Kit according to claim 31, characterized in that it contains either 1, 2 or 3 of the four possible deoxyribonucleoside triphosphates (dNTPs) or all four possible dNTPs for carrying out the chain termination polymerizations, one of these dNTPs being replaced by a chain termination deoxyribonucleoside triphosphate ,

33. Kit according to claim 32, characterized in that the chain termination deoxyribonucleoside triphosphate is a ddNTP, 3'0-methyl-NTP or 3 '-amino-NTP.

34. Kit according to claims 31 to 33, characterized in that the components for determining the elongate length comprise devices and means for carrying out an electrophoretic method, a mass spectrometric method or a fluorescence correlation spectroscopy.

35. Kit claims 31 to 34, characterized in that it additionally contains the components of a kit claims 25 to 29.

36. Kit according to claims 31 to 35, characterized in that it is a kit for the detection of prokaryotes, which contains two highly conserved sequences from the rRNA region as amplification primers.

37. Kit according to claim 36, characterized in that the amplification primers are the primers according to SEQ ID NO 1 and 2 or the primers according to SEQ ID NO 3 and 4.

38. Kit according to claims 36 and 37, characterized in that it contains at least one sequencing primer which hybridizes with a conserved region of the rRNA region.

39. Kit according to claim 38, characterized in that the sequencing primer (s) is / are selected from the group consisting of sequencing primers according to SEQ ID NO 5, 6, 7, 8, 9, 10 and 11.

40. Kit according to claim 39, characterized in that it contains a mixture of the sequencing primers according to SEQ ID NO 5 and 6, a mixture of the sequencing primers according to SEQ ID NO 7 to 9 and a mixture of the sequencing primers according to SEQ ID NO 10 and 11 in one or separate containers.

41. Kit according to claims 31 to 40, characterized in that it is a kit for the detection of sepsis pathogens.