Abstract:
The present invention relates to a method for detecting and/or identifying Gram-positive bacteria with high GC content, in particular mycobacteria. The method comprises a nucleic acid amplification reaction and a subsequent hybridization reaction with suitable primers and probes. The invention furthermore relates to oligonucleotides, compositions, solid phases and kits which may be used for carrying out the method of the invention.

Description:
This application is a 371 of PCT/EP02/03956 filed Apr. 9, 2002, which claims priority to German Application No. 101 21 505.3 filed May 2, 2001. 
     FIELD OF THE INVENTION 
     The present invention relates to the field of diagnostics of microorganisms, in particular to detection of Gram-positive bacteria with high GC content, such as mycobacteria, in clinical diagnostics. 
     BACKGROUND OF THE INVENTION 
     The detection of bacteria and their accurate identification play a very important role in clinical diagnostics. Thus, a particular pathogen in bacterial infections should be identified as quickly as possible in order to be able to initiate an appropriate treatment. 
     In this context, Gram-positive bacteria, in particular mycobacteria, play an important part. Currently more than 100 mycobacterial species are known which have been detected and differentiated from clinical material. Pathogenic mycobacterial species such as the  Mycobacterium tuberculosis  complex,  Mycobacterium intracellulare  and  Mycobacterium avium  are of particular importance here. 
     In medical routine diagnostics and in food analysis, bacteria have traditionally been identified via selective media and subsequent investigation of the biochemical properties. Frequently, however, it is not possible here to determine the exact species. Moreover, these studies are very time-consuming, and a pure culture must be present for more extensive studies. Highly complex and diverse groups of bacteria with difficult growth conditions in particular are difficult to access by traditional culture differentiation and/or slow down diagnostics considerably due to their slow growth. 
     In recent years, nucleic acid-based methods have increasingly been introduced in order to detect bacteria. Said methods comprise, for example, carrying out nucleic acid amplification reactions using species-specific primers. Detection is usually via gel electrophoresis or via immobilized probes in microtiter plates. However, such techniques are not suitable for detecting one or more out of a multiplicity of possible pathogenic organisms. One approach to this problem is an amplification reaction mixture containing a mixture of different species-specific amplification primers and corresponding probes and/or a primer pair complementary to a base section of a group of organisms. The species specificity is ensured here via the structure of the probes. 
     The ribosomal RNA (rRNA) or ribosomal DNA (rDNA) including its spacer structures has already been used as target sequence for amplification of bacteria-specific nucleic acid. The target sequence most frequently used diagnostically is 16S rRNA. It is the best represented sequence in the appropriate databases. However, due to the relatively conserved character, species-characteristic sequence structures are not always found. In contrast, the 16S-23S rDNA spacer region is highly variable within many species and well suited to identifying bacteria. It is also superior to the 16S rRNA with respect to its structural information content. However, these target regions have also only limited suitability for distinguishing between a large number of mycobacterial species, since their sequence variability is too high and thus the sensitivity to probes against said target region decreases. 
     Liesack et al. (1991) FEBS Lett. 281, 114-118 disclose the complete nucleotide sequence of  Mycobacterium leprae  23S and 5S rRNA and various target sequences for detection of bacteria. A probe of the  Helix  54 region for detecting  Mycobacterium leprae  was disclosed, inter alia. This, however, does not allow the conclusion that it is possible to distinguish between a very large number of different species of Gram-positive bacteria with high GC content, for example mycobacteria, on the basis of the  Helix  54 target region. 
     SUMMARY OF THE INVENTION 
     The present invention relates to a method for detecting and/or identifying Gram-positive bacteria with high GC content, according to which method first a nucleic acid amplification reaction is carried out and then a composition comprising the amplification product or a part thereof is hybridized with one or more probes. Finally, the degree of hybridization is determined. 
     Various reactions may be used as nucleic acid amplification reaction. Preference is given to using the polymerase chain reaction (PCR). The various designs of the PCR technique are known to the skilled worker, see, for example, Mullis (1990) Target amplification for DNA analysis by the polymerase chain reaction. Ann Biol Chem (Paris) 48(8), 579-582. Further amplification techniques which may be applied are nucleic acid strand-based amplification (NASBA), transcriptase mediated amplification (TMA), reverse transcriptase polymerase chain reaction (RT-PCR), Q-β replicase amplification (β-Q-Replicase) and single strand displacement amplification (SDA). NASBA and other transcription-based amplification methods are discussed in Chan and Fox, Reviews in Medical Microbiology (1999), 10(4), 185-196. 
     The nucleic acid amplification reaction is carried out using a sample composition. Said sample composition may be any composition which is suspected of containing bacteria, in particular Gram-positive bacteria with high GC content. It may be primary material, for example tracheal secretions, wound smears, blood etc., or cultures of microorganisms previously grown in liquid or on solid media. 
     According to the invention, the primer pair present in the nucleic acid amplification reaction is a first primer having any of the sequences SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7 or SEQ ID NO. 9 and a second primer having any of the sequences SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 or SEQ ID NO. 10. The sequences of the individual primers are depicted in  FIG. 1 . 
     In a preferred embodiment, any of the primer pairs having the sequences 
     SEQ ID NO. 1/SEQ ID NO. 2 (primer pair 1), 
     SEQ ID NO. 3/SEQ ID NO. 4 (primer pair 2), 
     SEQ ID NO. 5/SEQ ID NO. 6 (primer pair 3), 
     SEQ ID NO. 7/SEQ ID NO. 8 (primer pair 4) and 
     SEQ ID NO. 9/SEQ ID NO. 10 (primer pair 5) 
     is present in the nucleic acid amplification reaction. 
     Any of said primer pairs may be used for amplifying a nucleic acid region which comprises at least part of the  Helix  54 of 23S rDNA/rRNA of Gram-positive bacteria with high GC content. The primers may, in different combinations, amplify the  Helix  54 region of all G+C-rich Gram-positive bacteria. The primer SEQ ID NO. 8 ( FIG. 1 ), in particular, is capable of binding specifically only to nucleic acid of G+C-rich Gram-positive bacteria under suitable reaction conditions. The combination of primer SEQ ID NO. 3 ( FIG. 1 ) with SEQ ID NO. 2 ( FIG. 1 ) or with SEQ ID NO. 4 ( FIG. 1 ) amplifies  M. chelonae  complex,  M. kansasii, M. malmoense, M. tuberculosis  complex,  M. avium, M. scrophulaceum  and  M. interjectum  in a highly specific manner. 
     The skilled worker appreciates that it is possible, starting from the teaching of the present invention, to design primers which deviate slightly from the primers of the invention but which function nevertheless. Thus, primers which are extended or truncated by at least one, two or three nucleotides at the 5′ and/or 3′ end compared to the inventive primers having the sequences SEQ ID NO. 1 to 10 are also conceivable. Extensions or truncations at the 5′ end of the primers, in particular, may still provide functional primers which may be used according to the invention. Likewise, it is conceivable that individual or some nucleotides of a primer are replaced with other nucleotides, as long as the specificity of said primers and the melting temperature of said primers are not altered too much. It is evident to the skilled worker that, apart from the usual nucleotides A, G, C and T, modified nucleotides such as inosin etc. may also be applied. The teaching of the present invention makes such modifications possible, starting from the subject matter of the claims. 
     According to the invention, a composition comprising the amplification product or a part thereof is hybridized with one or more probes. The probes are oligonucleotides which have any of the sequences SEQ ID NO. 11 to 42 or the sequences complementary thereto.  FIGS. 2A and 2B  depict the sequences of the individual probes. Slight modifications of the probes are also conceivable, with negligible impairment of their functionality. The probes of the present invention may be extended and/or truncated by a few nucleotides, preferably by no more than three nucleotides, more preferably by no more than two nucleotides, most preferably by one nucleotide, at the 5′ end and/or the 3′ end. It is likewise possible to replace one or some nucleotides in the sequence of the probes of the invention with a different nucleotide, as long as hybridization to the target sequence is still possible. This includes the fact that in modifications the melting temperature of the modified probe does not deviate too much from the melting temperature of the original probe. The melting temperature is calculated according to the formula:
 
melting temperature=(number of  G/C  base pairs)·4° C.+
 
+(number of A/T base pairs)·2° C.
 
In principle, hybridization with a single specific probe can determine the bacterial species. However, it is also possible to hybridize the composition comprising the amplification product or a part thereof with more than one probe. This increases the meaningfulness of the method. The most accurate statement is possible if a hybridization is carried out with all probes having the sequences SEQ ID NO. 11 to 42 (or the sequences complementary thereto). In this case, an accurate profile is obtained and the bacterial species can be determined with great certainty. Thus, for example, probe SEQ ID NO. 23 ( FIG. 2A ) hybridizes with nucleic acid from the bacteria  M. intracellulare, M. scrophulaceum  and  M. interjectum. M. intracellulare , however, may be removed from  M. scrophulaceum  and  M. interjectum  by probe SEQ ID NO. 24 ( FIG. 2A ). Probe SEQ ID NO. 25 ( FIG. 2A ) hybridizes with nucleic acid from  M. celatum, M. chelonae  complex,  M. gordonae, M. kansasii, M. malmoense, M. tuberculosis  complex,  M. scrophulaceum  and  M. interjectum  and other mycobacterial species but, as far as we know, only with species of the genus mycobacteria. It removes, together with SEQ ID NO. 23 ( FIG. 2A ), the species  M. scrophulaceum  and  M. interjectum  from all other mycobacteria, according to current knowledge. This is, in a similar way, also true for probe SEQ ID NO. 24.
 
     The hybridization is usually carried out in such a way that either the composition comprising the amplification product or a part thereof or the probe is immobilized on a solid phase and contacted with the respective other hybridization partner. Possible solid phases are a large variety of materials, for example nylon, nitrocellulose, polystyrene, siliceous materials, etc. It is also possible to use a microtiter plate as solid phase. 
     Normally, at least one probe or at least one primer is labeled. A large variety of labels are possible such as, for example, fluorescent dyes, biotin or digoxigenin. Common fluorescent labels are fluorescein, FITC, cyanine dyes, etc. The labels are usually covalently linked to the oligonucleotides. Biotin and digoxigenin labels can be detected with suitable binding molecules after incubation, while a fluorescent label can be detected directly. A biotin-labeled oligonucleotide, for example, may be detected by contacting it with a solution containing streptavidin coupled to an enzyme, said enzyme, for example peroxidase or alkaline phosphatase, converting a substrate which generates a dye or produces chemiluminescence. Methods of this kind are known per se to the skilled worker. 
     In one embodiment of the invention, at least one of the primers used is labeled. Preferably, a plurality of the primers present in the reaction are labeled. If labeled primers are used, then the probes are usually not labeled. In this embodiment of the method, the probes are immobilized on a solid phase which is then contacted with the composition comprising the amplification product or a part thereof. This method is advantageous in that it is possible to immobilize more than one probe on the solid phase. Preference is given to immobilizing at least two probes, more preferably at least five probes, still more preferably at least ten probes, most preferably the probes having the sequences SEQ ID NO. 11 to 42 or the correspondingly complementary sequences, on the solid phase. Incubation of the amplification product or a part thereof with a solid phase with immobilized probes, which has been prepared in this way, can obtain, via a single hybridization step, information about hybridization of the amplification product with all immobilized probes. Therefore, the solid phase is preferably a microarray of immobilized probes on a solid phase. Such “DNA chips” allow immobilizing of a large number of different oligonucleotides on a small area. The solid phases suitable for DNA chips are preferably composed of siliceous materials such as glass etc. In this embodiment, the primer label is preferably a fluorescent label. The DNA chip may be rapidly analyzed by a scanning device, after incubation with the amplification product. Such devices are known to the skilled worker. A review of chip technology can be found in McGlennen (2001) Miniaturization technologies for molecular diagnostics, Clin Chem 47(3), 393-402. 
     In another embodiment, at least one of the probes has a label. In this case, the composition comprising the amplification product or a part thereof is usually immobilized on a solid phase and contacted with a composition comprising at least one probe selected from the group consisting of probes having the sequences SEQ ID NO. 11 to 42 and sequences complementary thereto. In this embodiment too, preference is given to carrying out a hybridization with more than one probe. For this purpose, it is possible to provide a plurality of solid phases on which the amplification product is immobilized. It is also possible, however, to immobilize small amounts of the amplification product on a plurality of spatially separated regions on a solid phase. These various spots are then contacted with in each case different probes (hybridization). 
     The procedure of the hybridization method is known per se to the skilled worker. Thus, after incubation with the solution which may contain the hybridization partner, the solid phases are usually subjected to stringent conditions in order to remove unspecifically bound nucleic acid molecules. The hybridization may be carried out in a conventional way on a nylon or nitrocellulose membrane, as described (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989). The principles mentioned therein can be transferred to further embodiments by the skilled worker. 
     According to the invention, the degree of hybridization is determined after hybridization. This is usually carried out by determining the amount of label bound to the solid phase. Detection reactions and detection methods of this kind are known per se to the skilled worker. 
     Another aspect of the invention is an oligonucleotide having any of the sequences SEQ ID NO. 1 to 42. The present invention likewise relates to an oligonucleotide having a sequence complementary to any of the sequences SEQ ID NO. 11 to 42. 
     The invention also relates to a composition comprising at least one such oligonucleotide. Preferably, the composition comprises a primer pair, with the first primer being selected from the group consisting of the primers having the sequences SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9 and the second primer being selected from the group consisting of the primers having the sequences SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10. More preferably, the composition comprises any of the primer pairs having the sequences 
     SEQ ID NO. 1/SEQ ID NO. 2 (primer pair 1), 
     SEQ ID NO. 3/SEQ ID NO. 4 (primer pair 2), 
     SEQ ID NO. 5/SEQ ID NO. 6 (primer pair 3), 
     SEQ ID NO. 7/SEQ ID NO. 8 (primer pair 4) and 
     SEQ ID NO. 9/SEQ ID NO. 10 (primer pair 5). 
     Another aspect of the invention is a solid phase on which at least one oligonucleotide having any of the sequences SEQ ID NO. 11 to 42 or a sequence complementary thereto is immobilized. If a plurality of oligonucleotides are immobilized, said oligonucleotides are spatially separated from one another on the solid phase. In a preferred embodiment, the oligonucleotides having the sequences SEQ ID NO. 11 to 42 or a sequence complementary thereto are immobilized on the solid phase. The solid phase is preferably designed as DNA chip. 
     The invention furthermore relates to a kit for detecting and/or identifying Gram-positive bacteria with high GC content, in particular mycobacteria, which kit comprises at least one oligonucleotide of the invention. In a particular embodiment, said kit comprises at least one primer pair, the first primer being selected from the group consisting of the primers having the sequences SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9 and the second primer being selected from the group consisting of the primers having the sequences SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10; and at least one oligonucleotide having any of the sequences SEQ ID NO. 11 to 42 or the sequences complementary thereto. More preferably, the kit comprises any of the primer pairs having the sequences 
     SEQ ID NO. 1/SEQ ID NO. 2 (primer pair 1), 
     SEQ ID NO. 3/SEQ ID NO. 4 (primer pair 2), 
     SEQ ID NO. 5/SEQ ID NO. 6 (primer pair 3), 
     SEQ ID NO. 7/SEQ ID NO. 8 (primer pair 4) and 
     SEQ ID NO. 9/SEQ ID NO. 10 (primer pair 5). 
     The preferred embodiments of the compositions, solid phases and kits of the present invention correspond to those of the method of the invention. 
     Finally, the invention also relates to the use of one or more of the oligonucleotides having the sequences SEQ ID NO. 1 to 10 as primers in a nucleic acid amplification reaction, in particular for detecting and/or identifying Gram-positive bacteria with high GC content. 
     The invention also relates to the use of one or more of the oligonucleotides having the sequences SEQ ID NO. 11 to 42 or sequences complementary thereto as probes in a hybridization reaction, in particular for detecting and/or identifying Gram-positive bacteria with high GC content. 
     The 23S rRNA/rDNA probes described herein and their application in the recognition and differentiation of bacteria of the phylogenetic branch of Gram-positive bacteria with high GC content are of great importance in medical microbiology. Normally said species can be differentiated by morphological or biochemical methods only with great difficulties and/or identification are slowed down considerably due to their slow growth. Conservative flanking regions make this gene region accessible to a nucleic acid amplification using a single set of primers (generic amplification primers). This makes it possible to use chip technology. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  depicts the nucleic acid sequences of the primers having the sequences SEQ ID NO. 1 to 10. 
         FIG. 2A  depicts a list of the probes having the sequences SEQ ID NO. 11 to 27 and the in each case related target organisms. 
         FIG. 2B  depicts the sequences of the probes having the sequences SEQ ID NO. 28 to 42 and the in each case related target organisms. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The following examples are intended to illustrate the invention in more detail. 
     Example 1 
     Isolation of DNA/RNA 
     Bacterial nucleic acid was obtained either from solid nutrient media, liquid media or from primary material, after appropriate preparation. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Bacterial species studied in Example 1 
               
             
          
           
               
                   
                 Species 
                 Abbreviation 
                 ATCC No. 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   Mycobacterium avium 
                 
                 M. avi 
                 25291 
               
               
                   
                 
                   Mycobacterium celatum 
                 
                 M. cel 
               
               
                   
                 
                   Mycobacterium chelonae 
                 
                 M. che 
                 14472 
               
               
                   
                 
                   Mycobacterium fortuitum 
                 
                 M. fo1 
                 6841 
               
               
                   
                 
                   Mycobacterium fortuitum 
                 
                 M. fo2 
               
               
                   
                 
                   Mycobacterium peregrinum 
                 
                 M. per 
               
               
                   
                 
                   Mycobacterium gordonae 
                 
                 M. gor 
                 14470 
               
               
                   
                 
                   Mycobacterium malmoense 
                 
                 M. mal 
                 29571 
               
               
                   
                 
                   Mycobacterium phlei 
                 
                 M. phl 
                 11758 
               
               
                   
                 
                   Mycobacterium tuberculosis 
                 
                 M. tub 
                 27294 
               
               
                   
                   
                   
                 25177 
               
               
                   
                 
                   Mycobacterium xenopi 
                 
                 M. xen 
                 19971 
               
               
                   
                 
                   Mycobacterium intracellulare 
                 
                 M. int 
                 13950 
               
               
                   
                 
                   Mycobacterium scrophulaceum 
                 
                 M. scr 
                 19981 
               
               
                   
                 
                   Mycobacterium interjectum 
                 
                 M. itm 
               
               
                   
                 
                   Mycobacterium kansasii 
                 
                 M. kan 
                 12478 
               
               
                   
                 
                   Mycobacterium marinum 
                 
                 M. mar 
                 827 
               
               
                   
                 
                   Escherichia coli 
                 
                 E. col 
               
               
                   
                 Human DNA 
                 hDNA 
               
               
                   
                 Negative control 
                 N-Kon 
               
               
                   
                   
               
             
          
         
       
     
     For this purpose, bacterial material was removed from solid media using a sterile inoculation loop and suspended in 300 μl of 10 mM Tris/HCl pH 7.5. From liquid cultures, 1 ml was removed, centrifuged in a bench top centrifuge at 13,000 rpm for 5 min and, after discarding the supernatant, resuspended in 300 μl of 10 mM Tris/HCl pH 7.5. Primary material was liquefied with acetyl cysteine and “decontaminated” with NaOH/SDS. The cell suspensions obtained in this way were incubated in a Thermomixer (Eppendorf, Hamburg, Germany) at 95° C. for 15 min, sonicated in an ultrasound bath (Bandelin) for 15 min and centrifuged in a bench top centrifuge at 13,000 rpm for 10 min. In each case, 5 μl of the supernatant were used in the amplification reaction. 
     Amplification: 
     All primers were commercially synthesized (Interactiva, Ulm, Germany). The PCR mixture contained 1× Taq buffer (Qiagen, Hilden, Germany), 1 mM of each primer, 200 μM dNTP (Roche) and 1U of Hotstar Taq polymerase (Qiagen, Hilden, Germany). The PCR amplification was carried out on a Thermocycler PE 9600 (ABI, Weiterstadt, Germany), with 95° C. for 15 min, 10 cycles with 95° C. for 30 s and 60° C. for 2 min and with 20 cycles with 95° C. for 10 s, 55° C. for 50 s and 70° C. for 30 s. 
     The NucliSens amplification kit (Organon Technika, Boxtel, The Netherlands) was used according to the manufacturer&#39;s instructions for RNA amplification using the NASBA technique:
     1. Preparation of the amplification mix: 8 μl “reagent sphere” in “reagent dilution” dissolved in buffer (contains the enzymes required for the reaction), 5 μl of KCl solution, final concentration 70 mM KCl and 2 μl of primer solution, final concentration 0.5 μM primer;   2. 5 μl of RNA solution added and incubated in a water bath at 41° C. for 60 min.   

     The DNA/RNA amplicon was detected either by using an agarose gel stained with ethidium bromide or by hybridization. 
     Detection of the Amplicons by Probe Hybridization: 
     All probes were biotinylated at the 5′ end in order to be able to detect target sequence/probe hybrids via reporter enzymes coupled to streptavidin. The probes used are oligonucleotide sequences SEQ ID NO. 11 to 27 (see  FIG. 2A ). 
     Absorbent paper (Blotting Papier GB002, Schleicher &amp; Schüll, Dassel, Germany) and a nylon membrane (Biodyne A, Pall, Portsmouth, England) were cut to the size of the blotting apparatus (Minifold Schleicher &amp; Schüll, Dassel, Germany) and soaked with 10×SSC. 250 μl of denaturation solution (50 mM NaOH; 1.5 M NaCl) were first introduced into the openings of the assembled apparatus and 20 μl of amplicon were added using a pipette. After applying a vacuum, the liquid was allowed to be sucked through completely. This was followed by rinsing with 10×SSC buffer. After the membrane had dried completely, it was fixed in a UV crosslinker (UV-Stratalinker 2400, Stratagene, La Jolla, USA) at 1200 Joule/cm 2  and washed with distilled water and dried. 
     All hybridizations were carried out in glass tubes in a hybridization oven (Hybaid Mini Oven MkII, MWG-Biotech, Ebersberg, Germany) at 50° C. The membrane coated with DNA/RNA amplicon was rolled up in the dry state and placed into a glass tube. The membrane was then incubated with continuous rotation with prewarmed hybridization buffer for 5 min. After adding 2 pmol of biotinylated probe, the hybridization reaction took place for one hour. Unbound or only partially bound probe was removed by incubating with stringent buffer at 50° C. for 30 min with a single exchange of the prewarmed stringent buffer. This was followed by adding blocking reagent and further incubating at 37° C. for 15 min. The hybrids were detected via a streptavidin-alkaline phosphatase conjugate either calorimetrically by adding NBT/BCIP or autoradiographically by spraying on a chemiluminescent substrate (Lumi-Phos 530, Cellmark Diagnostics, Abindon, England). For this purpose, streptavidin-alkaline phosphatase conjugate was added, followed by incubation at 37° C. for 30 min. The membrane was subsequently washed twice with substrate buffer for 15 min each. The membrane was then removed, Lumi-Phos reagent was sprayed on, followed by exposure of an X-ray film for 2 h. Alternatively, substrate buffer containing NBT/BCIP was added and the color was allowed to develop. 
     Solutions Used 
     10×SSC solution (standard saline citrate): 
     1.5 M NaCl, 0.15M trisodium citrate; 
     Hybridization buffer: 
     7% SDS (sodium dodecyl sulfate), 0.25 M phosphate buffer pH 7.5; 
     Stringent washing solution (stringent buffer): 
     3 M TMCL (tetramethylammonium chloride), 50 mM Tris/Cl, 2 mM EDTA, 0.1% SDS; 
     Solution for saturating the membrane binding sites: 
     5 g/l blocking reagent (Roche) in maleic acid buffer pH 7.5 (4.13 g of NaCl and 5.53 g of maleic acid in 500 ml of water, pH adjusted to 7.5 with 5 M NaOH); 
     Substrate buffer: 
     274 mM Tris/Cl pH 7.5, 68.6 mM Na 3  citrate, 200 mM NaCl, 27.4 mM MgCl 2 *6H 2 O; 
     BCIP: 
     50 mg/ml 5-bromo-4-chloro-3-indonyl phosphate, toluidinium salt in 100% dimethylformamide; 
     NBT: 
     75 mg/ml nitroblue tetrazolium salt in 70% dimethylformamide; 
     The autoradiograms were evaluated densitometrically. The 100% base value used was the amplicondot of the species from which the probe sequence had been derived. Controls which were always carried along as dots on the membrane were a sample to which water rather than nucleic acid solution had been added and a sample containing 100 ng of isolated human DNA. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Results of Example 1. The percentages of the densitometric 
               
               
                 evaluation are listed. The value of the probe homologous 
               
               
                 for the species was set to 100%. The probes used here 
               
               
                 may also have overlapping specificities. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Probe 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Taxon 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
               
               
                   
               
               
                 M. avi 
                 100.0 
                 100.0 
                 22.3 
                 13.8 
                 30.6 
                 13.9 
                 21.9 
                 13.2 
                 14.5 
               
               
                 M. cei 
                 11.1 
                 100.0 
                 100.0 
                 19.8 
                 22.5 
                 08.6 
                 11.3 
                 18.1 
                 10.2 
               
               
                 M. che 
                 14.0 
                 75.4 
                 21.1 
                 100.0 
                 11.2 
                 14.8 
                 10.4 
                 20.7 
                 12.9 
               
               
                 M. fo1 
                 19.1 
                 92.9 
                 11.1 
                 17.8 
                 100.0 
                 22.6 
                 40.7 
                 09.5 
                 15.0 
               
               
                 M. fo2 
                 11.3 
                 100.0 
                 12.6 
                 18.8 
                 27.6 
                 100.0 
                 34.0 
                 10.0 
                 11.4 
               
               
                 M. per 
                 17.3 
                 100.0 
                 24.8 
                 19.4 
                 14.2 
                 11.4 
                 100.0 
                 22.8 
                 14.6 
               
               
                 M. gor 
                 08.6 
                 100.0 
                 17.4 
                 21.4 
                 02.3 
                 10.9 
                 14.6 
                 100.0 
                 04.8 
               
               
                 M. mal 
                 08.4 
                 94.7 
                 12.8 
                 19.7 
                 09.6 
                 11.4 
                 14.2 
                 17.5 
                 100.0 
               
               
                 M. phl 
                 14.6 
                 100.0 
                 09.5 
                 04.6 
                 08.4 
                 05.5 
                 08.4 
                 08.1 
                 03.5 
               
               
                 M. tub 
                 17.8 
                 88.6 
                 11.6 
                 18.1 
                 20.1 
                 14.2 
                 12.8 
                 18.4 
                 14.8 
               
               
                 M. xen 
                 04.5 
                 100.0 
                 02.4 
                 03.7 
                 02.6 
                 01.8 
                 05.4 
                 01.2 
                 01.6 
               
               
                 M. int 
                 19.5 
                 100.0 
                 21.4 
                 19.4 
                 28.3 
                 25.4 
                 19.2 
                 29.1 
                 24.6 
               
               
                 M. scr 
                 20.1 
                 87.3 
                 19.4 
                 24.5 
                 27.4 
                 20.3 
                 20.8 
                 20.1 
                 22.9 
               
               
                 M. itm 
                 21.5 
                 100.0 
                 17.1 
                 22.1 
                 28.4 
                 25.4 
                 20.1 
                 20.9 
                 29.3 
               
               
                 M. kan 
                 09.1 
                 79.3 
                 08.5 
                 09.4 
                 08.1 
                 09.9 
                 10.2 
                 08.8 
                 07.4 
               
               
                 M. mar 
                 18.6 
                 100.0 
                 14.3 
                 11.5 
                 20.4 
                 21.2 
                 11.1 
                 10.9 
                 17.7 
               
               
                 E. col 
                 07.5 
                 08.0 
                 07.4 
                 07.2 
                 06.8 
                 06.3 
                 07.4 
                 16.9 
                 06.8 
               
               
                 hDNA 
                 0.1 
                 0.2 
                 0.02 
                 0.04 
                 0.05 
                 0.1 
                 0.06 
                 0.1 
                 0.05 
               
               
                 N-kon 
                 0.04 
                 0.02 
                 0.01 
                 0.01 
                 0.02 
                 0.02 
                 0.1 
                 0.1 
                 0.06 
               
               
                   
               
             
          
           
               
                 Probe 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Taxon 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
                 17 
               
               
                   
               
               
                 M. avi 
                 24.1 
                 10.4 
                 09.1 
                 31.1 
                 80.3 
                 34.1 
                 16.2 
                 14.6 
               
               
                 M. cei 
                 09.5 
                 10.4 
                 08.9 
                 75.9 
                 19.6 
                 18.7 
                 10.7 
                 12.3 
               
               
                 M. che 
                 08.4 
                 07.8 
                 12.3 
                 17.6 
                 19.3 
                 82.0 
                 23.9 
                 10.2 
               
               
                 M. fo1 
                 08.9 
                 09.4 
                 11.3 
                 19.2 
                 11.9 
                 19.6 
                 10.8 
                 06.4 
               
               
                 M. fo2 
                 14.0 
                 09.1 
                 15.5 
                 18.7 
                 14.6 
                 22.5 
                 14.9 
                 09.9 
               
               
                 M. per 
                 24.8 
                 21.4 
                 19.8 
                 19.2 
                 10.2 
                 21.6 
                 28.0 
                 14.6 
               
               
                 M. gor 
                 14.5 
                 17.5 
                 14.1 
                 20.1 
                 01.8 
                 88.2 
                 09.9 
                 10.4 
               
               
                 M. mal 
                 10.2 
                 10.8 
                 18.4 
                 12.8 
                 17.8 
                 74.3 
                 09.7 
                 12.4 
               
               
                 M. phl 
                 100.0 
                 01.9 
                 09.6 
                 11.8 
                 09.8 
                 04.1 
                 05.3 
                 02.4 
               
               
                 M. tub 
                 14.9 
                 100.0 
                 16.2 
                 21.4 
                 14.6 
                 82.4 
                 24.5 
                 10.0 
               
               
                 M. xen 
                 01.6 
                 01.7 
                 100.0 
                 01.4 
                 02.0 
                 02.1 
                 01.4 
                 01.8 
               
               
                 M. int 
                 20.0 
                 34.7 
                 18.9 
                 100.0 
                 100.0 
                 41.4 
                 15.0 
                 19.7 
               
               
                 M. scr 
                 17.5 
                 32.4 
                 19.2 
                 100.0 
                 29.1 
                 78.9 
                 15.5 
                 17.8 
               
               
                 M. itm 
                 19.4 
                 38.4 
                 24.6 
                 100.0 
                 34.0 
                 100.0 
                 11.8 
                 10.8 
               
               
                 M. kan 
                 14.1 
                 07.4 
                 04.6 
                 18.4 
                 09.8 
                 92.9 
                 100.0 
                 07.1 
               
               
                 M. mar 
                 19.5 
                 22.8 
                 12.3 
                 10.8 
                 08.4 
                 10.5 
                 14.8 
                 100 
               
               
                 E. col 
                 07.9 
                 07.8 
                 08.4 
                 07.2 
                 07.4 
                 07.4 
                 07.8 
                 06.1 
               
               
                 hDNA 
                 0.01 
                 0.2 
                 0.5 
                 0.04 
                 0.1 
                 0.05 
                 0.02 
                 0.08 
               
               
                 N-kon 
                 0.04 
                 0.09 
                 0.2 
                 0.06 
                 0.06 
                 0.01 
                 0.01 
                 0.04 
               
               
                   
               
             
          
         
       
     
     The methods described here may be used to identify and differentiate the corresponding bacteria either from primary material (e.g. tracheal secretions, wound smears, blood, and the like) or from bacterial liquid or solid media. In the case of material which is not primarily sterile, the accompanying flora which is not acid-resistant must be removed beforehand by common decontamination methods. This applies both to the setting-up of the culture and to the preparation of the primary material. 
     Example 2 
     Using the methods according to Example 1, further samples containing Gram-positive bacteria with high GC content were studied. The bacterial species studied were: 
     
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Bacterial species studied in Example 2 
               
             
          
           
               
                   
                 ATCC No. 
               
               
                   
                   
               
             
          
           
               
                 
                   Corynebacterium amycolatum 
                 
                 C. amy 
                   
               
               
                 
                   Corynebacterium callunae 
                 
                 C. cal 
               
               
                 
                   Corynebacterium diphteriae gravis 
                 
                 C. dpg 
               
               
                 
                   Corynebacterium glutamicum 
                 
                 C. glu 
               
               
                 CDC Coryneform Group G 
                 C. grG 
               
               
                 
                   Corynebacterium jeikeium 
                 
                 C. jei 
               
               
                 
                   Corynebacterium matrucchotii 
                 
                 C. mat 
               
               
                 
                   Corynebacterium minutissimun 
                 
                 C. min 
               
               
                 
                   Corynebacterium pseudodiphteriticum 
                 
                 C. pdp 
               
               
                 
                   Corynebacterium pseudotuberculosis 
                 
                 C. ptb 
               
               
                 
                   Corynebacterium ulcerans 
                 
                 C. ulc 
               
               
                 
                   Corynebacterium striatum 
                 
                 C. str 
               
               
                 
                   Corynebacterium xerosis 
                 
                 C. xer 
                 373 
               
               
                 
                   Nocardia asteroides 
                 
                 N. ast 
               
               
                 
                   Rhodococcus equi 
                 
                 R. equ 
               
               
                 
                   Tsukamurella paurometabolum 
                 
                 T. prm 
               
               
                 
                   Escherichia coli 
                 
                 E. col 
               
               
                 Human DNA 
                 hDNA 
               
               
                 Negative Control 
                 N-Kon 
               
               
                   
               
             
          
         
       
     
     The probes used are oligonucleotides having the sequences SEQ ID NO. 28 to 42 (see  FIG. 2B ). 
     The evaluation was likewise carried out as described in Example 1. The results are summarized in Table 4. 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Results of Example 2. The percentages of the densitometric 
               
               
                 evaluation are listed. The value of the probe homologous 
               
               
                 for the species was set to 100%. The probes used here 
               
               
                 may also have overlapping specificities. 
               
             
          
           
               
                 Probe 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Taxon 
                 18 
                 19 
                 20 
                 21 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
                 29 
                 30 
                 31 
                 32 
               
               
                   
               
             
          
           
               
                 C. amy 
                 100.0 
                 09.4 
                 11.4 
                 04.9 
                 24.2 
                 14.8 
                 11.0 
                 18.4 
                 21.1 
                 11.8 
                 14.2 
                 15.5 
                 19.7 
                 09.4 
                 08.1 
               
               
                 C. cal 
                 19.5 
                 100.0 
                 11.7 
                 18.8 
                 20.8 
                 20.0 
                 11.8 
                 14.6 
                 14.5 
                 18.4 
                 19.2 
                 12.1 
                 11.5 
                 18.6 
                 18.4 
               
               
                 C. dpg 
                 14.4 
                 18.5 
                 100.0 
                 12.7 
                 17.6 
                 13.7 
                 14.4 
                 19.4 
                 20.0 
                 26.1 
                 09.7 
                 11.1 
                 17.8 
                 13.2 
                 17.1 
               
               
                 C. glu 
                 19.5 
                 11.8 
                 17.9 
                 100.0 
                 12.4 
                 17.6 
                 19.2 
                 14.6 
                 11.8 
                 19.8 
                 19.9 
                 14.8 
                 09.6 
                 18.6 
                 14.8 
               
               
                 C. grG 
                 21.9 
                 12.8 
                 18.2 
                 18.3 
                 100.0 
                 15.8 
                 20.3 
                 11.6 
                 14.4 
                 15.9 
                 22.1 
                 13.5 
                 17.6 
                 11.2 
                 15.9 
               
               
                 C. jei 
                 09.8 
                 19.4 
                 12.5 
                 18.4 
                 12.8 
                 100.0 
                 18.8 
                 12.6 
                 14.6 
                 12.4 
                 11.1 
                 18.1 
                 19.7 
                 17.4 
                 14.8 
               
               
                 C. mat 
                 15.4 
                 13.6 
                 13.8 
                 12.7 
                 17.6 
                 16.5 
                 100.0 
                 10.9 
                 11.7 
                 11.2 
                 13.5 
                 15.8 
                 17.2 
                 12.5 
                 19.7 
               
               
                 C. min 
                 18.5 
                 14.6 
                 13.8 
                 17.6 
                 12.6 
                 12.1 
                 20.5 
                 100.0 
                 15.4 
                 16.4 
                 17.6 
                 17.4 
                 12.5 
                 04.6 
                 22.9 
               
               
                 C. pdp 
                 15.2 
                 16.6 
                 17.1 
                 11.2 
                 20.1 
                 08.6 
                 15.4 
                 14.9 
                 100.0 
                 08.9 
                 18.4 
                 18.4 
                 13.8 
                 14.8 
                 20.1 
               
               
                 C. ptb 
                 14.6 
                 14.6 
                 18.6 
                 17.6 
                 14.2 
                 14.3 
                 18.5 
                 14.8 
                 18.7 
                 100.0 
                 23.4 
                 11.3 
                 10.9 
                 17.3 
                 28.1 
               
               
                 C. ulc 
                 16.4 
                 16.9 
                 21.4 
                 18.6 
                 15.4 
                 13.5 
                 17.2 
                 17.5 
                 17.4 
                 100.0 
                 24.9 
                 12.3 
                 10.0 
                 19.1 
                 27.4 
               
               
                 C. str 
                 05.6 
                 19.4 
                 14.6 
                 12.4 
                 18.4 
                 16.9 
                 18.3 
                 12.1 
                 14.1 
                 18.2 
                 100.0 
                 10.2 
                 10.9 
                 19.5 
                 20.4 
               
               
                 C. xer 
                 14.5 
                 12.6 
                 19.4 
                 15.6 
                 14.6 
                 15.4 
                 12.5 
                 42.1 
                 8 
                 16.4 
                 13.8 
                 100.0 
                 14.3 
                 12.8 
                 11.3 
               
               
                 N. ast 
                 07.4 
                 08.1 
                 09.2 
                 10.3 
                 04.2 
                 03.6 
                 08.8 
                 04.6 
                 09.5 
                 10.8 
                 02.6 
                 11.5 
                 100.0 
                 08.5 
                 05.2 
               
               
                 R. equ 
                 03.4 
                 06.6 
                 08.5 
                 08.4 
                 01.6 
                 08.8 
                 06.6 
                 04.8 
                 08.1 
                 05.1 
                 08.2 
                 06.3 
                 04.3 
                 100.0 
                 05.2 
               
               
                 T. prm 
                 04.3 
                 02.6 
                 06.5 
                 02.9 
                 09.4 
                 06.6 
                 06.3 
                 08.2 
                 08.6 
                 01.6 
                 05.4 
                 08.4 
                 04.2 
                 03.6 
                 100.0 
               
               
                 E. col 
                 08.5 
                 05.3 
                 07.1 
                 05.5 
                 04.2 
                 04.2 
                 09.1 
                 07.2 
                 01.4 
                 05.4 
                 04.1 
                 02.2 
                 08.5 
                 05.1 
                 04.2 
               
               
                 hDNA 
                 0.1 
                 0.03 
                 0.05 
                 0.08 
                 0.09 
                 0.1 
                 0.1 
                 0.04 
                 0.05 
                 0.08 
                 0.1 
                 0.09 
                 0.08 
                 0.1 
                 0.04 
               
               
                 N-kon 
                 0.02 
                 0.02 
                 0.01 
                 0.03 
                 0.01 
                 0.02 
                 0.1 
                 0.504 
                 0.08 
                 0.02 
                 0.01 
                 0.1 
                 0.08 
                 0.08 
                 0.02