WO1995013395A1 - Specific gene probes and methods for quantitative detection of methicillin-resistant staphylococci - Google Patents

Specific gene probes and methods for quantitative detection of methicillin-resistant staphylococci Download PDF

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WO1995013395A1
WO1995013395A1 PCT/EP1994/003553 EP9403553W WO9513395A1 WO 1995013395 A1 WO1995013395 A1 WO 1995013395A1 EP 9403553 W EP9403553 W EP 9403553W WO 9513395 A1 WO9513395 A1 WO 9513395A1
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dna
amplification
gene
hybridization
methicillin
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PCT/EP1994/003553
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German (de)
French (fr)
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Wolfgang Springer
Rainer Endermann
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Bayer Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • Methicillin-resistant staphylococci are important nosocomial pathogens. You are responsible for severe postoperative wound infections, bacteremia and infections caused by foreign materials introduced into the body, e.g. Catheters, going out.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSE Staphylococcus epidermidis
  • MRSA / M SE are resistant to all beta-lactam antibiotics such as penicillins, cephalosporins, penems and carbapenems. 80% of the MRSA / MRSE strains are also resistant to other classes of antibiotics such as macrolides, aminoglycosides and quinolones.
  • methicillin-resistant staphylococci have an additional so-called mec A gene which codes for the penicillin binding protein PBP2a.
  • This PBP2a has an extremely low affinity for all ß-lactam antibiotics and therefore, as a transpeptidase, enables cell wall synthesis in the presence of ß-lactam concentrations that inhibit all other PBPs (Neu, Science 257, 1064 (1992)).
  • this mec A gene is present in all methicillin-resistant staphylococci and the homology between the various mec A genes is very high, it was a good idea to use this gene to perform rapid DNA diagnostics with specific gene probes combined with known DNA or To develop RNA application methods.
  • the present invention describes specific oligonucleotide and polynucleotide probes and their use for the rapid detection of methine-resistant staphylococci directly in the clinical sample material.
  • the gene probes were produced from the gene sequence of the mec A gene by chemical synthesis (oligonucleotide probes) or PCR cloning (polynucleotide probes).
  • the preferred gene probes were selected from a range that
  • a) is specific for methicillin-resistant S. aureus and S. epidermidis
  • the application of parts of the mec A gene was carried out using specific primers from the coding and non-coding strand of the mec A gene while simultaneously labeling the amplification product with digoxigenin dUTP using known amplification methods, preferably the PCR-DNA amplification method (EP 200 362) or the HAS RNA amplification method (EP 427 074).
  • known amplification methods preferably the PCR-DNA amplification method (EP 200 362) or the HAS RNA amplification method (EP 427 074).
  • the hybridization complex is separated with streptavidin-coupled magnetizable particles.
  • methicin-resistant staphylococci The amount or the presence of methicin-resistant staphylococci is carried out by a chemiluminescence test with antidigoxigenin antibodies which are combined with alk. Phosphatase coupled (PCR) or by DNA / RNA
  • Antibodies with alk. Phosphatase are coupled (HAS amplification).
  • the evaluation can be used semi-quantitatively to determine the amount of methicin-resistant staphylococcal in the clinical sample material.
  • Gene probe diagnostics is a fast, specific and highly sensitive method that enables early detection of the pathogen at the DNA / RNA level.
  • the technique can be carried out directly in the test material without in vitro cultivation. It is based on the DNA / RNA hybridization technique, ie the specific in vitro binding of complementary single-stranded nucleic acid to form Watson-Crick base pairs.
  • the DNA / DNA or DNA / RNA double strands formed are also referred to as DNA hybrids.
  • Complementary sequence-specific gene probes are used to detect the specific DNA or RNA of a pathogen by the hybridization reaction.
  • These gene probes are either short, chemically synthesized oligonucleotide probes with a length of 10 to 50 nucleotides or DNA / RNA fragments of 0.5 to 10 kb, which were produced by recombinant genetic engineering.
  • the gene probes can be photochemically (N. Dattagupta, et al., Biochem. 177, 85, 1989) or enzymatically by nick translation (Rigby, PWJ et al., J. Mol. Biol. 113, 237, 1977) or random primed techniques (Feinberg and Vogelstein, Anal. Biochem. 132, 6, 1983) can be provided with a radioactive or non-radioactive label. Suitable for this are labels with 32 P NTPs or non-radioactive labels with digoxigenin-dUTP, biotin-dUTP or direct labeling with enzymes such as alk. Phosphatase or Horseradish Peroxidase.
  • the nucleic acids are first separated into single strands by denaturation (heat or alkali treatment) and then very specifically with one another under stringent conditions which are achieved by temperature, ionic strength of the buffer and organic solvents hybridizes.
  • the gene probe only binds to complementary sequences of the DNA or RNA to be detected.
  • This hybridization reaction can be carried out in various test formats, for example as solid-phase hybridization to a carrier such as, for example, nitrocellulose-coupled target DNA or gene probe, or as a liquid hybridization.
  • the evaluation takes place via the labeling of the gene probe with a reporter molecule as listed above or, as in the reversed phase hybridization system shown here, via the target DNA which is labeled with digoxigenin-dUTP during the amplification and the gene probe which is labeled with biotin for binding to magnetizable particles.
  • the hybridization complex of target DNA and labeled gene probe is determined quantitatively after removal of non-hybridized DNA via the reporter molecule used.
  • This read out can be done directly with fluorescence labeling or radioactive labeling or indirectly by enzyme tests and immunological methods with antibody conjugates, the enzymes such as the alk. Contain phosphatase and then allow a color reaction or chemiluminescence reaction.
  • test sensitivity with this gene probe diagnosis is in the range from 10 5 to 10 6 germs based on the detection of single genes.
  • An increase in test sensitivity can be achieved by combining it with DNA or RNA amplification techniques such as the PCR (EP 200 362). LCR (EP 320 308), NASBA (EP 329 822), Qß (PCT 87/06270) or HAS technology (EP 427 074) can be achieved. With these techniques, up to a 10 9- fold multiplication of the DNA to be detected can be achieved. The combination of amplification and hybridization makes it possible to detect individual DNA molecules.
  • the new gene probes were developed from gene areas of the mec A gene which have specific and particularly strong hybridization signals for methicillin-resistant staphylococci and which detect both methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), but do not result in a signal with methicillin-sensitive staph.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSE Staphylococcus epidermidis
  • the construction of oligonucleotide probes and polynucleotide probes for methicillin-resistant staphylococci is described in the invention.
  • GenBank and EMBL nucleotide sequence databases no homologies to known gene probes (Ligozzi, M., et al., Antimicrob. Agents Chemother. 35, 575-578, 1991) (U-Gene Research, WO 9108305) or primers for the amplification (EP 0 526 876,
  • a method for using these gene probes in hybridization tests is also described, in which a specific hybridization with target DNA of methicin-resistant staphylococci takes place. Furthermore, this invention describes the combination of these hybridization methods with amplification techniques, in particular the hairpin amplification method (EP 427 074), by means of which a very strong improvement in test sensitivity is achieved.
  • the nucleotide sequence of the mec A gene from Staphylococcus aureus and Staphylococcus epidermidis was used for the selection of suitable specific oligonucleotide probes (Ryffel et al., Gene 94, 137 (1990) Song et al., FEBS Letters 221, 167 (1987) sequences which are conserved in the mec A gene of MRSA / MRSE but have no homology to the known PBPs
  • the preferred oligonucleotide probe is described in the sequence listing SEQ ID No. 1.
  • this oligonucleotide probe solid phase hybridization tests with, for example, digoxigenin end-labeled probes or Liquid hybridization tests with, for example, photodigoxigenin-labeled genomic DNA and biotin-labeled oligonucleotide probes with which the hybridization complex is then separated with the aid of streptavidin-coupled magnetizable particles are carried out.
  • the disadvantage of this method is the relatively low detection limit of 10 5 pathogens If lower pathogen concentrations occur, this procedure is not sufficient for early diagnosis.
  • the described gene probe technique was therefore combined with DNA and RNA amplification methods such as e.g. the PCR technique (EP 200 362, EP 201 184) and hairpin amplification technique (EP 427 074).
  • the amplification products often contain, depending on the process conditions, such as the annealing temperature, primer and enzyme concentration, primer sequence and MgCL2 concentration and used
  • Polymerase by means of primer mismatching, non-specific by-products which pretend false positive results when the amplification products are stained in the gel or with fluorescence labeling during the amplification.
  • the selected oligonucleotide probe was chemically synthesized using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.
  • the polynucleotide probe was carried out by PCR cloning using a SureCloneTM ligation kit from Pharmacia Biochemicals. Was obtained, cloned in the pUC 18 vector, a polynucleotide probe of 467 NuMeotiden with the NuMeotide sequence described in the sequence listing SEQ ID No 2.
  • the 467 bp probe was isolated from the vector by restriction enzyme cleavage, agarose gel electrophoresis and electroelution and then labeled using standard labeling methods (random prime, 3-end group labeling) with, for example, biotin-d-UTP.
  • the probe was produced directly by PCR synthesis with primers 3 and 4 (SEQ EB No 9 and 10) with the simultaneous incorporation of biotin-d-UTP.
  • this gene probe can be used directly for the hybridization of genomic DNA (slot blot hybridization, reversed phase liquid hybridization or amplified mec A DNA).
  • primers were used for the amplification of the mec A gene or parts thereof (e.g. EP 527 628 or EP 526 876).
  • the primer 1 sequence listing SEQ ID No 3 and primer 2 sequence listing SEQ ID No 4 are particularly well suited for the specific detection of methicillin-resistant staphylococci. With the genomic DNA of methicillin-sensitive staphylococci, they produce no amplification product visible in the agarose gel.
  • Genomic DNA from methicin-resistant staphylococci results in a strong amplification band that hybridizes well with the oligonucleotide probe or the polyMeotide probe, thereby achieving very good test sensitivities of ⁇ 10 germs / ml sample material.
  • dNTPs deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate
  • Digoxigenin-dUTP can be incorporated into the amplification product. This allows the amplification product to be treated with an antidigoxigenin antibody, e.g. alk.
  • Phosphatase coupled contains a chemiluminescence test with AMPPD as a substrate or a dye test with bromine-chloro-indolyl phosphate and nitro blue tetrazolium.
  • fluorescence-labeled NuMeoside triphosphates such as Incorporate fluorescein dUTP or coumarin dUTPs into the amplification product and identify the amplification product with much higher sensitivity than with ethidium bromide staining.
  • a T7 / T3 hairpin oligonucleotide which, in addition to the hairpin sequence, contains a sequence corresponding to the oligonucleotide sequences from the mec A oligonucleotide probe (SEQ ID No 6-8).
  • RNA transcripts are then produced with T7 / T3 polymerase, which hybridize with, for example, biotinylated capture gene probes (SEQ ID No 5) and can be separated from the reaction solution with magnetized particles coated on streptavidin.
  • the DNA / RNA complex can then be detected by DNA / RNA specific antibodies (EP 339 686), which with alk. Phosphatase are coupled.
  • the MRSA / MRSE can be detected directly via their genomic DNA using the gene probes described in the invention.
  • the evaluation can be carried out quantitatively via slot blot hybridization or reversed phase liquid hybridization.
  • the test sensitivity with approx. 10 5 cells / ml sample material is relatively low and therefore only of limited suitability for the early detection of infections with these pathogens.
  • the amplification of the mec A gene by the primers described in the invention which are suitable for various read-out methods with fluorescence, biotin, digoxigenin or enzymes such as alk. Phosphatase or Horse Radish Peroxidase can be marbled.
  • a possible read out method is the staining of the amplification product separated by agarose gel electrophoresis with intercalating agents such as ethidium bromide. Another possibility is the incorporation of fluorescence-mari ned NuMeosidtriphosphaten in the DNA or RNA amplification product. This leads to a significant improvement in test sensitivity.
  • the most reliable and most sensitive method is the method of hybridizing the amplification products with the MRSA / MRSE-specific gene probes described in the invention. This method also allows a quantification of the MRSA / MRSE in mini sample material ( Figure 1).
  • digoxigenin-dUTP is incorporated during the amplification and use of biotin-marbled gene probes
  • the hybridization complex of streptavidin-coated magnetized particles can be separated and when using antidigoxigenin antibodies which are combined with alk.
  • Phosphatase are coupled, with AMPPD as a substrate semi-quantitatively via chemiluminescence. example 1
  • oligonucleotide probes and starter oligonucleotides were chemically synthesized using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.
  • the following NuMeotide sequences were synthesized:
  • Oligonucleotide probe SEQ ID No 1
  • PCR primer 2 SEQ ID No 4 HAS capture probe, 5'-biotinylated: SEQ ID No 5
  • T3 hairpin oligo with mec A oligonucleotide sequence SEQ ID No 6
  • T7 hairpin oligo with mec A oligonucleotide sequence SEQ ID No 7
  • SP6 hairpin oligo with mec A oligonucleotide sequence SEQ ID No 8
  • PCR primer 3 SEQ ID No 9
  • PCR primer 4 SEQ ID No 10
  • the oligonucleotide probe was marmered using the method of Bollum, The enzymes Boyer ed, Vol 10, p 145, Academic Press New York, at the 3 'end with Biotin-dUTP. End group marching was not radioactive with digoxigenin-dUTP (Chang, L.M.S., Bollum T.J., J. Biol. Chem. 246, 909, 1971).
  • the PolynuMeotidsonde was obtained by PCR cloning with a SureCloneTM Ligati on Kit from Pharmacia Biochemicals.
  • 100 ng of genomic DNA from MRSA / MRSE, 200 ⁇ mol dNTPs, 1.9 mM MgCl 2.2 ⁇ mol primer 3 (SEQIDNO9) and primer 4 (SEQEDNO10) and PCR buffer were used.
  • the DNA per ZyMus was denatured for 1 minute at 94 ° C. and then the primer annealing was carried out for 2 minutes at 50 ° C. Subsequently, the primer extension was carried out at 72 ° C. for 2 minutes.
  • the reaction was treated at 72 ° C. for 20 minutes and then the PCR cloning was carried out immediately.
  • the dATP ends at the 3 'end of the PCR product were removed by the 3'-5' exonuase reactivity of the Klenows fragment.
  • the PCR fragments were then phosphorylated with T4 PolynuMeotidMnase and after a phenol / chloroform treatment and MicroSpin column chromatography the PCR product Blunt End ligated into the pUC 18 VeMor dephosphorilized with bovine alkaline phosphatase. This was followed by the transformation and selection of PCR clones using standard methods (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).
  • the 467 bp probe isolated from the polymer sequences of the VeMor was marmered by random prime standard methods with Biotin-d-UTP.
  • the gene probe was produced directly by PCR synthesis with primer 3 and primer 4 with simultaneous incorporation of 0.2 ⁇ mol biotin-d-UTP and used in the genetic tests and example 6.
  • the amplification of the target DNA was carried out after the polymerase chain reaction (EP 200 362; 201 184).
  • PCR reaction 1-1000 pg of genomic DNA from methicin-resistant staphylococci, 2 ⁇ mol of primer 1 SEQ ID No 3 and primer 2 SEQ ID No 4, 2.5 units of Taq polymerase from Cetus / PerMn-Elmer and 200 ⁇ mol of dNTPS each were used in a total batch of 100 ⁇ l PCR buffer (50 mM KC1, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl 2 , and 0.01% gelatin.
  • PCR buffer 50 mM KC1, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl 2 , and 0.01% gelatin.
  • the amplification was carried out in a PCR processor from Cetus / PerMn
  • a PCR processor from Cetus / PerMn
  • an additional 0.15 ⁇ mol digoxigenin-d-UTP was used in the PCR to mar the PCR product.
  • the DNA was initially melted for 2 minutes, 30 seconds at 94 ° C, then the DNA was denatured for 1 minute at 94 ° C per cycle, the primer annealing for 1 minute 30 seconds at 50 ° C and 1 minute 30
  • the primer extension was carried out for seconds at 72 ° C. After 35 cycles, a 10 minute extension was then carried out at 72 ° C. and the batches were cooled at 4 ° C.
  • RNA amplification For RNA amplification, a sandwich hybridization was carried out using a 5'biotinylated capture probe (SEQ ID No 5) of the genomic DNA from staphylococci and the 5'phosphorylated hairpin oligonucleotide SEQ ID No 6-8, the hybridization complex being coupled to streptavidin-coated magnetizable particles was. After ligation of capture probe and hairpin oligonucleotide, the transcription amplification of the T7 / T3 hairpin is carried out using the corresponding RNA polymerase.
  • SEQ ID No 5 5'biotinylated capture probe
  • SEQ ID No 6-8 the 5'phosphorylated hairpin oligonucleotide SEQ ID No 6-8
  • 500 fmol capture oligo were with 500 fmol hairpinoligo and 10 to 100 amol target.
  • -DNA boiled in 50 ⁇ l T10E1 buffer for 5 minutes.
  • 50 ul target hybridization buffer was added and then incubated at 52 ° C for 10 minutes.
  • 100 ⁇ l of Dynal Streptavidin particles were added, incubated for 10 minutes at room temperature and then magnetically separated and the supernatant was discarded.
  • 10 ⁇ l of ligase premix buffer were added and ligated at 37 ° C. for 15 minutes.
  • 25 ml transcription buffer (IVT-MIX) with T7 RNA polymerase were added. After 2.5 hours at 37 ° C the RNA amplification was finished.
  • TlOEl buffer 10MM Tris / HCl; pH 8, ImM EDTA Target 10 ml
  • intercalating agents such as e.g. Ethidium bromide or during the amplification fluorescence NuMeotidtriphosphate such as e.g. Fluorescent dUTP or coumarin dUTP incorporated.
  • Biotin-dUTP or digoxigenin-dUTP can also be used, and alk.
  • Phosphatase a dye readout can be performed.
  • Appropriately labeled primers can also be used if the sensitivity is lower.
  • the preferred method was the incorporation of coumarin dUTP because the best test sensitivity was achieved.
  • the amplification product was applied to a 0.8% agarose gel and electrophoresed at 70 mA for 30 minutes.
  • the fluorescence signals from methicine-sensitive and methicin-resistant staphylococci were evaluated under a UV transilluminator direM.
  • PCR PCR
  • LCR LCR
  • NASBA EP 329 822
  • Qß PCT 87/06270
  • HAS EP 427 074
  • liquid hybridization tests were carried out as reversed phase tests with 100 ng 3'-biotinylated oligonucleotide probe and amplified DNA according to Example 3 in a volume of 50 ⁇ l.
  • the BlocMng reaction and antibody reaction for detection of hybridization via chemiluminescence was then carried out.
  • the beads loaded with DNA were treated 1 ⁇ with 150 ⁇ l washing buffer (0.1 M maleic acid, 0.1 MNaCl pH 7.5, 0.3% Tween 20) and, after separating and pipetting off the washing buffer, 400 ⁇ l buffer 2 (0 , 1M maleic acid; 0.15M NaCl, pH 7.5; 1% BlocMng reagent (Boehringer)) added.
  • Example 6 B describes a simplified alternative chemiluminescence test method to Example 6 A which can be carried out in 1 hour with the same test sensitivity and can be automated.
  • liquid hybridization tests were carried out as reversed phase tests with 100 ng 3'-biotinylated gene probe and amplified DNA according to Example 3 in a volume of 50 ⁇ l.
  • the coupled hybridization complex was separated with the beads , the remaining liquid is pipetted off and washed once with buffer B (0.1 SSC; 0.1% SDS) once.
  • the BlocMng reaction and antibody reaction were then carried out to detect the hybridization via chemiluminescence.
  • the beads loaded with DNA were added 1x with 500 ⁇ l buffer 2 (0.1M maleic acid; 0.15M NaCl pH7.5; 1% Bloc ng reagent (Boehringer)) After 5 minutes of incubation at room temperature, the mixture was separated, pipetted off and 250 ⁇ l of antibody conjugate solution (AK 1: 2500 in buffer 2) were added and incubated for 10 minutes at room temperature, then separated, pipetted off and treated with 500 ⁇ l wash buffer 2x 30 seconds, lx 2 minutes The movement was then incubated with 100 ⁇ l detection solution with AMPPD 1: 100 in buffer 3 for 10 minutes at 37 ° C. in a water bath, then the chemiluminescence was measured in the luminescence photometer at 477 nm (Lumacounter from Lumac).
  • 500 ⁇ l buffer 2 0.1M maleic acid; 0.15M NaCl pH7.5; 1% Bloc ng reagent (Boehringer)
  • the MRSA / MRSE-specific RNA transcripts (approx. 0.5 pmol) were hybridized with the capture oligo (4 pmol) and 10 ⁇ l 3 ⁇ transcription buffer 2 in a total volume of 30 ⁇ l.
  • the mixture was denatured for 2 minutes at 98 ° C., then hybridized for 10 minutes at 57 ° C. and then 70 ⁇ l H 2 O were added.
  • the mixture was incubated at room temperature for 10 minutes, magnetically separated and the supernatant was discarded. Then it was washed 3 times with 200 ⁇ l washing buffer and separated. The mixture was then washed twice with 200 ⁇ l of antibody binding buffer.
  • Binding buffer [0.1M Tris.HCl, 0.1M NaCl, 0.1% BSA, 0.1% Tween]
  • the sample material infected with staphylococci for example infected blood, was centrifuged at 8000 rpm for 5 to 10 minutes, the supernatant was pipetted off and discarded.
  • the sediment (approx. 180 to 200 ⁇ l) was mixed with 50 ⁇ l TE with 15% sucrose, 20 ⁇ l lysozyme + lysostaphin (10+ 5 mg / ml in bidist. H 2 O) was added and incubated for 15 minutes at 37 ° C . Further processing was carried out using a Diagen Quiamp DNA kit. After addition of 25 ⁇ l Proteinase K and 235 ⁇ l AL buffer, the mixture was mixed and treated at 70 ° C. for 10 minutes.
  • the MRSA / MRSE DNA was isolated from the mini sample material according to the method described in Example 8.
  • the staphylococcal DNA lysate was then amplified using suitable amplification methods as described in Example 5 with MRSA / MRSE-specific oligonucleotide primers.
  • the amplified nucleic acid was then hybridized with the oligonucleotide probe SEQ ID No 1 or the polynucleotide probe SEQ ID No 2 and the specific hybridization complex of amplified staphylococcal nucleic acid and gene probe DNA which developed under stringent conditions was separated with magnetizable particles from Dynal and as in the example 6 quantified by chemiluminescence read out.
  • mec A gene-specific hybridization signals were still easily detected in a concentration of 10 1 germs.
  • Example 10 Quantitative detection of MRSA / MRSE MRSA / MRSE nucleic acid were isolated from clinical sample material, for example blood, as in Example 8. The amplification was carried out as described in Example 3. The number of cycles was limited to 25 cycles in order to achieve a good correlation between the number of cells and the chemiluminescence signal in the clinically relevant range from 10 3 to 10 germs. In addition to the clinical samples, samples with MRSA MRSE DNA corresponding to the cell numbers from 10 6 to 10 ° cells were amplified in parallel in 500 ng blood DNA and analyzed in the chemiluminescence test (Example 6). FIG.
  • FIG. 1 shows the chemiluminescence signals of 10 6 to 10 MRSA MRSE in the blood in comparison to test samples with 10 3 and 10 2 germs amplified in parallel, which had been detected microbiologically.
  • the diagram shows that the chemiluminescence signals from the cell number DNA standard (10 3 -10 2 ) can be compared very well with the chemiluminescence signals of the test samples with 10 3 and 10 2 germs and the chemiluminescence test for quantifying the MRSA / MRSE eg can be used in the blood.
  • Figure 1 shows a chemiluminescence test for the semi-quantitative detection of MRSA.
  • the amplification was carried out as in Example 3, the number of cycles being limited to 25 cycles in order to achieve a good correlation between cell number and chemiluminescence signal in the clinically relevant range from 1000 to 10 germs / ml of sample material.
  • the diagram shows the chemiluminescence signals of a cell standard of 10 6 to 10 MRSA in blood in comparison to test samples with 1000 and 100 germs, which were also amplified in parallel, and which had been detected microbiologically. From the diagram it can be seen that the chemiluminescence signals from the cell DNA standard (10 2 -10 3 ) can be compared very well with the chemiluminescence signals of the test samples with 1000 and 100 germs and the chemiluminescence test for the semi-quantitative determination of MRSA, for example in Blood can be used.
  • SEQUENCE LOG SEQUENCE LOG
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • ORGANISM Staphylococcus aureus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Staphylococcus aureus
  • AAACAAGTTA TAAAATCGAT GGTAAAGGTT GGCAAAAAGA TAAATCTTGG GGTGGTTACA 300
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • ORGANISM Staphylococcus aureus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Staphylococcus aureus
  • ORGANISM Staphylococcus aureus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCCATTTATG TATGGCATGA GTAACG 26
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Staphylococcus aureus / T3 Phage
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Staphylococcus aureus / T7 phage
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Staphylococcus aureus / SP6 phage
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Staphylococcus aureus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Staphylococcus aureus

Abstract

The invention describes new gene probes and methods for quantitative determination of methicillin-resistant staphylococci.

Description

Spezifische Gensonden und Verfahren zum quantitativen Nachweis von methiciUinresistenten StaphylococcenSpecific gene probes and methods for the quantitative detection of methicin-resistant staphylococci
Seit Mitte der siebziger Jahre nahm der Anteil an methiciUinresistenten Stämmen bei Staphylococcenmfektionen von wenigen Prozenten bis auf 70 % deutlich zu. Während das Auftreten dieser Stämme zunächst nur auf große Universitätskliniken beschränkt war, findet man heute diese resistenten Stämme auch immer häufiger in Krankenhäusern der Grundversorgung (Paul et al., Abstr. 23 ICAAC (1991)).Since the mid-1970s, the proportion of methicin-resistant strains in staphylococcal infections has increased significantly from a few percent to 70%. While the occurrence of these strains was initially restricted to large university clinics, these resistant strains are now increasingly found in primary care hospitals (Paul et al., Abstr. 23 ICAAC (1991)).
Methicillinresistente Staphylococcen sind wichtige nosokomiale Erreger. Sie sind verantwortlich für schwere postoperative Wundinfektionen, Bakteriämien und Infektionen, die von in den Körper eingebrachten Fremdmaterialien, z.B. Kathedern, ausgehen.Methicillin-resistant staphylococci are important nosocomial pathogens. You are responsible for severe postoperative wound infections, bacteremia and infections caused by foreign materials introduced into the body, e.g. Catheters, going out.
Von besonderer klinischer Bedeutung sind methicillinresistente Staphylococcus aureus (MRSA) und Staphylococcus epidermidis (MRSE) während Staphylococcus hämolyticus Infektionen nur gelegentlich auftreten.Of particular clinical importance are methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), while Staphylococcus hemolyticus infections occur only occasionally.
MRSA/M SE sind gegen alle ß-Laktamantibiotika, wie Penicilline, Cephalosporine, Peneme und Carbapeneme resistent. 80 % der MRSA/MRSE Stämme besitzen auch Resistenzen gegen andere Antibiotikaklassen wie Makrolide, Aminoglykoside und Chinolone.MRSA / M SE are resistant to all beta-lactam antibiotics such as penicillins, cephalosporins, penems and carbapenems. 80% of the MRSA / MRSE strains are also resistant to other classes of antibiotics such as macrolides, aminoglycosides and quinolones.
Es ist deshalb besonders wichtig möglichst frühzeitig eine Therapie mit dem wirk¬ samen Antibiotikum einzuleiten. Die Alternativen für die Therapie von Infektionen mit methiciUinresistenten Staphylococcen sind auf wenige Antibiotika beschränkt. Vankomycin ist das Antibiotikum der Wahl. Ein anderes Glycopeptidantibiotikum, Teicoplanin, wurde kürzlich erst eingeführt. Eine weitere Therapiemöglichkeit ist die Kombination von Antibiotika wie Ciprofloxacin mit Rifampicin. Die klassische Diagnostik der MRSA/MRSE ist zeit- und arbeitaufwendig und er¬ fordert die in vitro Kultivierung der Stämme auf Platten mit anschließender physio¬ logischer Identifizierung mit Hilfe von Testsystemen wie z.B. "api staph" (BioMerieux) und Bestimmung der Antibiotikaresistenzen durch Reihenverdünnungs- teste (MHK- Werte) oder Agardiffusionsteste (Hemmhofteste).It is therefore particularly important to initiate therapy with the effective antibiotic as early as possible. The alternatives for the therapy of infections with methicin-resistant staphylococci are limited to a few antibiotics. Vankomycin is the antibiotic of choice. Another glycopeptide antibiotic, teicoplanin, has only recently been launched. Another therapeutic option is the combination of antibiotics such as ciprofloxacin with rifampicin. The classic diagnosis of MRSA / MRSE is time-consuming and labor-intensive and requires the in vitro cultivation of the strains on plates with subsequent physical identification using test systems such as "api staph" (BioMerieux) and determination of antibiotic resistance by serial dilution. tests (MIC values) or agar diffusion tests (inhibitory tests).
Methicillinresistente Staphylococcen besitzen im Gegensatz zu den in sensitiven Stämmen vorhandenen Penicillin Bindeprotein-Genen ein zusätzliches sogenanntes mec A Gen, das für das Penicillin-Bindeprotein PBP2a codiert. Dieses PBP2a hat eine äußerst geringe Affinität für alle ß-Lactamantibiotika und ermöglicht deshalb als Transpeptidase die Zellwandsynthese in Gegenwart von ß-Lactamkonzentrationen, die alle anderen PBPs inhibieren (Neu, Science 257, 1064 (1992)).In contrast to the penicillin binding protein genes found in sensitive strains, methicillin-resistant staphylococci have an additional so-called mec A gene which codes for the penicillin binding protein PBP2a. This PBP2a has an extremely low affinity for all ß-lactam antibiotics and therefore, as a transpeptidase, enables cell wall synthesis in the presence of ß-lactam concentrations that inhibit all other PBPs (Neu, Science 257, 1064 (1992)).
Da dieses mec A Gen in allen Methicillin-resistenten Staphylococcen vorhanden ist und die Homologie zwischen den verschiedenen mec A Genen sehr hoch ist, bot es sich an auf der Basis dieses Gens eine DNA-Schnell-Diagnostik mit spezifischen Gensonden kombiniert mit bekannten DNA- oder RNA- Applikationsverfahren zu entwickeln.Since this mec A gene is present in all methicillin-resistant staphylococci and the homology between the various mec A genes is very high, it was a good idea to use this gene to perform rapid DNA diagnostics with specific gene probes combined with known DNA or To develop RNA application methods.
Die vorliegende Erfindung beschreibt spezifische Oligonukleotid- und Polynukleotid- sonden und ihre Verwendung zum schnellen Nachweis von methiciUinresistenten Staphylococcen direkt im klinischen Probenmaterial.The present invention describes specific oligonucleotide and polynucleotide probes and their use for the rapid detection of methine-resistant staphylococci directly in the clinical sample material.
1. Die Gensonden wurden aus der Gensequenz des mec A Gens durch chemi¬ sche Synthese (Oligonukleotid-Sonden) oder PCR-Klonierung (Polynukleo- tid-Sonden) hergestellt.1. The gene probes were produced from the gene sequence of the mec A gene by chemical synthesis (oligonucleotide probes) or PCR cloning (polynucleotide probes).
Die bevorzugten Gensonden wurden aus einem Bereich ausgewählt, derThe preferred gene probes were selected from a range that
a) spezifisch für methicillinresistente S. aureus und S. epidermidis ista) is specific for methicillin-resistant S. aureus and S. epidermidis
b) in anderen Penicillin Bindeprotein-Genen nicht vorkommt undb) does not occur in other penicillin binding protein genes and
c) alle methiciUinresistenten Staphylococcen mit MHK-Werten zwischen 4 bis 128 μg/ml eindeutig erfaßt. 2. Die genomische DNA von methiciUinresistenten Staphylococcen wurde durch an sich bekannte Verfahren, die für Staphylococcus adaptiert wurden, isoliert.c) all methicin-resistant staphylococci with MIC values between 4 to 128 μg / ml clearly recorded. 2. The genomic DNA of methicin-resistant staphylococci was isolated by methods known per se, which were adapted for Staphylococcus.
3. Die Applikation von Teilen des mec A Gens wurde durch spezifische Primer aus dem kodierenden und nicht kodierenden Strang des mec A Gens unter gleichzeitiger Markierung des Amplifikationsproduktes mit Digoxi- genin-dUTP mit Hilfe bekannter Amplifikationsmethoden, bevorzugt der PCR-DNA-Amplifιkationsmethode(EP 200 362) oder der HAS-RNA-Ampli- fikationsmethode (EP 427 074) durchgeführt.3. The application of parts of the mec A gene was carried out using specific primers from the coding and non-coding strand of the mec A gene while simultaneously labeling the amplification product with digoxigenin dUTP using known amplification methods, preferably the PCR-DNA amplification method (EP 200 362) or the HAS RNA amplification method (EP 427 074).
4. Der Nachweis des spezifischen Amplifikationsproduktes erfolgte durch Hybri- disierung des Amplifikationsproduktes mit den obengenannten, biotinylierten4. The detection of the specific amplification product was carried out by hybridizing the amplification product with the above-mentioned, biotinylated
Gensonden. Dadurch wird mit einem Nachweis von 10 Keimen eine signifikant höhere Sensitivität erreicht als durch direkten Nachweis des Am¬ plifikationsproduktes im Gel, bei dem maximal 103 Keime nachgewiesen werden.Gene probes. In this way, a detection of 10 germs achieves a significantly higher sensitivity than by direct detection of the amplification product in the gel, in which a maximum of 10 3 germs are detected.
5. Der Hybridisierungskomplex wird mit Streptavidin gekoppelten magneti- sierbaren Partikeln separiert.5. The hybridization complex is separated with streptavidin-coupled magnetizable particles.
6. Die Auswertung des gebildeten Hybridisierungskomplex als Maß für die6. The evaluation of the hybridization complex formed as a measure of the
Menge oder das Vorhandensein von methiciUinresistenten Staphylococcen erfolgt durch einen Chemilumineszenztest mit Antidigoxigenin-Antikörpern die mit alk. Phosphatase gekoppelt sind (PCR) oder durch DNA/RNA-The amount or the presence of methicin-resistant staphylococci is carried out by a chemiluminescence test with antidigoxigenin antibodies which are combined with alk. Phosphatase coupled (PCR) or by DNA / RNA
Antikörper, die mit alk. Phosphatase gekoppelt sind (HAS-Amplifikation).Antibodies with alk. Phosphatase are coupled (HAS amplification).
Die Auswertung kann bei Verwendung eines externen mitamplifizierten Staphylococcen-DNA-Standard semi quantitativ zur Ermittlung der Menge an methiciUinresistenten Staphylococcen im klinischen Probenmaterial eingesetzt werden.When using an external co-amplified staphylococcal DNA standard, the evaluation can be used semi-quantitatively to determine the amount of methicin-resistant staphylococcal in the clinical sample material.
Die Gensonden-Diagnostik insbesondere in Verbindung mit Amplifikationstechniken ist eine schnelle, spezifische und hochempfindliche Methode, die eine Früherkennung der Erreger auf DNA/RNA Ebene ermöglicht. Die Technik kann direkt ohne in vitro Kultivierung im Untersuchungsmaterial durchgeführt werden. Sie basiert auf der DNA/RNA Hybridisierungstechnik, d.h. der spezifischen in vitro Bindung von komplementärer Einzel strang-Nukl einsäure unter Bildung von Watson-Crick- Basenpaaren. Die gebildeten DNA/DNA oder DNA/RNA Doppelstränge werden auch als DNA-Hybride bezeichnet. Zur Detektion der spezifischen DNA oder RNA eines Erregers durch die Hybridisierungsreaktion werden komplementäre sequenzspezifische Gensonden verwendet. Diese Gensonden sind entweder kurze, chemisch synthetisierte Oligonukleotidsonden mit einer Länge von 10 bis 50 Nukleotiden oder DNA/RNA Fragmente von 0,5 bis 10 kb, die durch rekombinante Gentechniken hergestellt wurden.Gene probe diagnostics, especially in conjunction with amplification techniques, is a fast, specific and highly sensitive method that enables early detection of the pathogen at the DNA / RNA level. The technique can be carried out directly in the test material without in vitro cultivation. It is based on the DNA / RNA hybridization technique, ie the specific in vitro binding of complementary single-stranded nucleic acid to form Watson-Crick base pairs. The DNA / DNA or DNA / RNA double strands formed are also referred to as DNA hybrids. Complementary sequence-specific gene probes are used to detect the specific DNA or RNA of a pathogen by the hybridization reaction. These gene probes are either short, chemically synthesized oligonucleotide probes with a length of 10 to 50 nucleotides or DNA / RNA fragments of 0.5 to 10 kb, which were produced by recombinant genetic engineering.
Die Gensonden können photochemisch (N. Dattagupta, et al., Biochem. 177, 85, 1989) oder enzymatisch durch nick Translation (Rigby, P.W.J. et al., J. Mol. Biol. 113, 237, 1977) oder Random Primed Techniken (Feinberg und Vogelstein, Anal. Biochem. 132, 6, 1983) mit einer radioaktiven oder nicht radioaktiven Markierung versehen werden. Geeignet sind hierfür Markierungen mit 32P NTPs oder nicht radioaktive Markierungen mit Digoxigenin-dUTP, Biotin-dUTP oder direkte Markierung mit Enzymen wie alk. Phosphatase oder Horseradish Peroxidase.The gene probes can be photochemically (N. Dattagupta, et al., Biochem. 177, 85, 1989) or enzymatically by nick translation (Rigby, PWJ et al., J. Mol. Biol. 113, 237, 1977) or random primed techniques (Feinberg and Vogelstein, Anal. Biochem. 132, 6, 1983) can be provided with a radioactive or non-radioactive label. Suitable for this are labels with 32 P NTPs or non-radioactive labels with digoxigenin-dUTP, biotin-dUTP or direct labeling with enzymes such as alk. Phosphatase or Horseradish Peroxidase.
Für die spezifische Hybridisierung zwischen der nachzuweisenden Nukleinsäure des Erregers und der erregerspezifischen Gensonde werden die Nukleinsäuren zunächst durch Denaturierung (Hitze oder Alkalibehandlung) in Einzelstränge getrennt und dann unter stringenten Bedingungen, die durch Temperatur, Ionenstärke der Puffer und organische Lösungsmittel erreicht werden, ganz spezifisch miteinander hybridisiert. Bei geeigneten Hybridisierungsbedingungen bindet die Gensonde nur an komplementäre Sequenzen der nachzuweisenden DNA oder RNA. Diese Hybridisierungsreaktion kann in verschiedenen Testformaten z.B. als Fest- phasenhybridisierung an einen Träger wie z.B. Nitrozellulose gekoppelter Target- DNA oder Gensonde oder als Flüssighybridisierung durchgeführt werden. Die Aus¬ wertung (Read Out) erfolgt über die Markierung der Gensonde mit einem Reportermolekül wie oben aufgeführt oder wie in dem hier dargestellten Reversed Phase Hybridisierungssystem über die Target-DNA, die während der Amplifikation mit Digoxigenin-dUTP markiert wird und die Gensonde, die zur Bindung an magnetisierbare Partikel mit Biotin markiert wird. Der Hybridisierungskomplex aus Target-DNA und markierter Gensonde wird nach Entfernen von nicht hybridisierter DNA über das verwendete Reportermolekül quantitativ bestimmt. Dieser Read Out kann direkt erfolgen bei Fluoreszenz-Markierung oder radioaktiver Markierung oder indirekt durch Enzymteste und immunologische Verfahren mit Antikörperkonjugaten, die Enzyme wie die alk. Phosphatase enthalten und dann eine Farbreaktion oder Chemilumineszenz-Reaktion ermöglichen.For the specific hybridization between the nucleic acid of the pathogen to be detected and the pathogen-specific gene probe, the nucleic acids are first separated into single strands by denaturation (heat or alkali treatment) and then very specifically with one another under stringent conditions which are achieved by temperature, ionic strength of the buffer and organic solvents hybridizes. With suitable hybridization conditions, the gene probe only binds to complementary sequences of the DNA or RNA to be detected. This hybridization reaction can be carried out in various test formats, for example as solid-phase hybridization to a carrier such as, for example, nitrocellulose-coupled target DNA or gene probe, or as a liquid hybridization. The evaluation (read out) takes place via the labeling of the gene probe with a reporter molecule as listed above or, as in the reversed phase hybridization system shown here, via the target DNA which is labeled with digoxigenin-dUTP during the amplification and the gene probe which is labeled with biotin for binding to magnetizable particles. The hybridization complex of target DNA and labeled gene probe is determined quantitatively after removal of non-hybridized DNA via the reporter molecule used. This read out can be done directly with fluorescence labeling or radioactive labeling or indirectly by enzyme tests and immunological methods with antibody conjugates, the enzymes such as the alk. Contain phosphatase and then allow a color reaction or chemiluminescence reaction.
Die Testsensitivität mit dieser Gensonden-Diagnostik liegt im Bereich von 105 bis 106 Keimen auf der Basis der Detektion von Einzelgenen. Eine Erhöhung der Test¬ sensitivität kann durch die Kombination mit DNA oder RNA-Amplifikations- techniken ie derPCR(EP 200 362). LCR (EP 320 308), NASBA (EP 329 822), Qß (PCT 87/06270) oder HAS-Technik (EP 427 074) erreicht werden. Mit diesen Techniken kann bis zu einer 109-fachen Multiplikation der nachzuweisenden DNA erzielt werden. Durch die Kombination von Amplifikation und Hybridisierung wird so die Detektion von einzelnen DNA-Molekülen möglich.The test sensitivity with this gene probe diagnosis is in the range from 10 5 to 10 6 germs based on the detection of single genes. An increase in test sensitivity can be achieved by combining it with DNA or RNA amplification techniques such as the PCR (EP 200 362). LCR (EP 320 308), NASBA (EP 329 822), Qß (PCT 87/06270) or HAS technology (EP 427 074) can be achieved. With these techniques, up to a 10 9- fold multiplication of the DNA to be detected can be achieved. The combination of amplification and hybridization makes it possible to detect individual DNA molecules.
Da bei Infektionen im Blut Keimzahlen von 10 -10 Keimen/ml auftreten, bietet sich mit dieser Technologie die Möglichkeit einer frühzeitigen Erkennung von methiciUinresistenten Infektionsverläufen.Since there are bacterial counts of 10 -10 germs / ml in infections in the blood, this technology offers the possibility of early detection of methicin-resistant infection courses.
In der vorliegenden Erfindung werden neue spezifische und besonders sensitive Gensonden für methicillinresistente Staphylococcen beschrieben.In the present invention, new specific and particularly sensitive gene probes for methicillin-resistant staphylococci are described.
Die neuen Gensonden wurden aus Genbereichen des mec A Gens entwickelt, die spezifische und besonders starke Hybridisierungssignale für methicillinresistente Staphylococcen aufweisen und sowohl methicillinresistente Staphylococcus aureus (MRSA) und Staphylococcus epidermidis (MRSE) erfassen, aber kein Signal mit methicillinsensitiven Staphylococcen ergeben. Die Konstruktion von Oligonukleotid- sonden und Polynukleotidsonden für methicillinresistente Staphylococcen wird in der Erfindung beschrieben. In GenBank und EMBL Nukleotidsequenz-Datenbanken wurden keine Homologien zu bekannten Gensonden (Ligozzi, M., et al., Antimicrob. Agents Chemother. 35, 575-578, 1991) (U-Gene Research, WO 9108305) oder Primern für die Amplifikation (EP 0 526 876, EP 0 527 628) gefunden.The new gene probes were developed from gene areas of the mec A gene which have specific and particularly strong hybridization signals for methicillin-resistant staphylococci and which detect both methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), but do not result in a signal with methicillin-sensitive staph. The construction of oligonucleotide probes and polynucleotide probes for methicillin-resistant staphylococci is described in the invention. In GenBank and EMBL nucleotide sequence databases no homologies to known gene probes (Ligozzi, M., et al., Antimicrob. Agents Chemother. 35, 575-578, 1991) (U-Gene Research, WO 9108305) or primers for the amplification (EP 0 526 876, EP 0 527 628) found.
Es wird darüber hinaus ein Verfahren zum Einsatz dieser Gensonden in Hybridisie- rungstesten beschrieben, bei denen eine spezifische Hybridisierung mit Target-DNA von methiciUinresistenten Staphylococcen erfolgt. Weiterhin wird in dieser Erfindung die Kombination dieser Hybridisierungsverfahren mit Amplifikationstechniken, insbesondere der Hairpinamplifikationsmethode (EP 427 074), beschrieben, durch die eine sehr starke Verbesserung der Testsensitivität erzielt wird.A method for using these gene probes in hybridization tests is also described, in which a specific hybridization with target DNA of methicin-resistant staphylococci takes place. Furthermore, this invention describes the combination of these hybridization methods with amplification techniques, in particular the hairpin amplification method (EP 427 074), by means of which a very strong improvement in test sensitivity is achieved.
Es wurde auch der Einsatz dieser Gensonden und Testverfahren zum Nachweis von methiciUinresistenten Staphylococcen in klinischem Probenmaterial beschrieben. Ein großer Vorteil dieses Verfahrens ist darüber hinaus die Möglichkeit einer semiquantitativen Bestimmung der Menge an methiciUinresistenten Infektionskeimen in dem Probenmaterial.The use of these gene probes and test methods to detect methicin-resistant staphylococci in clinical specimens has also been described. Another great advantage of this method is the possibility of a semi-quantitative determination of the amount of methicin-resistant infection germs in the sample material.
Auswahl und Synthese von OligonukleotidsondenSelection and synthesis of oligonucleotide probes
Für die Auswahl von geeigneten spezifischen Oligonukleotidsonden wurde die Nukleotidsequenz des mec A Gens von Staphylococcus aureus und Staphylococcus epidermidis herangezogen (Ryffel et al., Gene 94, 137 (1990) Song et al., FEBS Letters 221, 167 (1987). Ausgewählt wurden solche Sequenzen, die konserviert im mec A Gen von MRSA/MRSE vorkommen, aber keine Homologie zu den bekannten PBPs aufweisen. Die bevorzugte Oligonukleotidsonde ist in dem Sequenzprotokoll SEQ ID No 1 beschrieben. Mit dieser Oligonukleotidsonde können Festphasen- Hybridisierungsteste mit z.B. Digoxigenin endmarkierten Sonden oder Flüssighybridisierungsteste mit z.B. Photodigoxigenin markierter genomischer DNA und biotinmarkierten Oligonukleotidsonden mit denen dann der Hybridisierungskomplex mit Hilfe von Streptavidin gekoppelten magnetisierbaren Partikeln separiert werden, durchgeführt werden. Der Nachteil dieser Verfahren ist die relativ geringe Nachweisgrenze von 105 Erregern. Da im Frühstadium der Infektion weitaus geringere Erregerkonzentrationen vorkommen, reicht dieses Ver- fahren für eine Frühdiagnose nicht aus.The nucleotide sequence of the mec A gene from Staphylococcus aureus and Staphylococcus epidermidis was used for the selection of suitable specific oligonucleotide probes (Ryffel et al., Gene 94, 137 (1990) Song et al., FEBS Letters 221, 167 (1987) sequences which are conserved in the mec A gene of MRSA / MRSE but have no homology to the known PBPs The preferred oligonucleotide probe is described in the sequence listing SEQ ID No. 1. With this oligonucleotide probe, solid phase hybridization tests with, for example, digoxigenin end-labeled probes or Liquid hybridization tests with, for example, photodigoxigenin-labeled genomic DNA and biotin-labeled oligonucleotide probes with which the hybridization complex is then separated with the aid of streptavidin-coupled magnetizable particles are carried out.The disadvantage of this method is the relatively low detection limit of 10 5 pathogens If lower pathogen concentrations occur, this procedure is not sufficient for early diagnosis.
In der vorliegenden Erfindung wurde deshalb die beschriebene Gensondentechnik kombiniert mit DNA- und RNA-Amplifikationsmethoden wie z.B. der PCR-Technik (EP 200 362, EP 201 184) und Hairpin-Amplifikationstechnik (EP 427 074).In the present invention, the described gene probe technique was therefore combined with DNA and RNA amplification methods such as e.g. the PCR technique (EP 200 362, EP 201 184) and hairpin amplification technique (EP 427 074).
Das Verfahren, bei dem zunächst das mec A Gen oder Teile davon aus der genomischen DNA amplifiziert werden und dann anschließend das Amplifikations- produkt spezifisch mit der Oligonukleotidsonde hybridisiert, hat gegenüber der reinen Amplifikationsdiagnostik folgende Vorteile:The method in which the mec A gene or parts thereof are first amplified from the genomic DNA and then subsequently the amplification Product hybridized specifically with the oligonucleotide probe has the following advantages over pure amplification diagnostics:
1. Die Amplifikationsprodukte enthalten häufig in Abhängigkeit von den Verfahrensbedingungen wie der Annealingtemperatur, Primer und Enzym- konzentration, Primersequenz und MgCL2-Konzentration und verwendeten1. The amplification products often contain, depending on the process conditions, such as the annealing temperature, primer and enzyme concentration, primer sequence and MgCL2 concentration and used
Polymerase, durch Primer-Mismatching unspezifische Nebenprodukte, die bei Anfärbung der Amplifikationsprodukte im Gel oder bei Fluoreszenz- Markierung während der Amplifikation falsch positive Ergebnisse vor¬ spiegeln.Polymerase, by means of primer mismatching, non-specific by-products which pretend false positive results when the amplification products are stained in the gel or with fluorescence labeling during the amplification.
2. Hinzu kommt die deutlich geringere Testsensitivität bei direkter Auswertung der PCR-Produkte, die bei 103 Keimen liegt, während in Kombination mit der Gensondenhybridisierung und einem Chemilumineszenz-Read Out ein Nachweis von 10 Keimen pro ml Probenmaterial problemlos gelingt.2. In addition, there is the significantly lower test sensitivity in the direct evaluation of the PCR products, which is 10 3 germs, while in combination with the gene probe hybridization and a chemiluminescence read-out, 10 germs per ml of sample material can be successfully detected.
Die chemische Synthese der ausgewählten Oligonukleotidsonde erfolgte nach der Phosphoramiditmethode von S.L. Beaucage und M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.The selected oligonucleotide probe was chemically synthesized using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.
Konstruktion der PolynukleotidsondeConstruction of the polynucleotide probe
Die Polynukleotidsonde wurde durch PCR-Klonierung mit einem SureCloneTM Ligation Kit der Firma Pharmacia Biochemicals durchgeführt. Erhalten wurde, Moniert in den pUC 18 Vektor, eine PolynuWeotidsonde von 467 NuMeotiden mit der NuMeotidsequenz beschrieben im Sequenzprotokoll SEQ ID No 2.The polynucleotide probe was carried out by PCR cloning using a SureCloneTM ligation kit from Pharmacia Biochemicals. Was obtained, cloned in the pUC 18 vector, a polynucleotide probe of 467 NuMeotiden with the NuMeotide sequence described in the sequence listing SEQ ID No 2.
Die 467 bp Sonde wurde aus dem Vektor durch Restriktionsenzymspaltung, Agarose- gelelektrophorese und Elektoelution isoliert und dann durch Standard-Markie¬ rungsmethoden (random prime, 3Εndgruppenmarkierung) mit z.B. Biotin-d-UTP markiert. Alternativ wurde die Sonde direkt durch PCR-Synthese mit Primer 3 und 4 (SEQ EB No 9 und 10) unter gleichzeitigem Einbau von Biotin-d-UTP hergestellt. Diese Gensonde kann wie die Oligonukleotidsonde direkt zur Hybridisierung von genomischer DNA (Slot Blot Hybridisierung, Reversed Phase Flüssighybridisierung oder amplifizierter mec A DNA) eingesetzt werden.The 467 bp probe was isolated from the vector by restriction enzyme cleavage, agarose gel electrophoresis and electroelution and then labeled using standard labeling methods (random prime, 3-end group labeling) with, for example, biotin-d-UTP. Alternatively, the probe was produced directly by PCR synthesis with primers 3 and 4 (SEQ EB No 9 and 10) with the simultaneous incorporation of biotin-d-UTP. Like the oligonucleotide probe, this gene probe can be used directly for the hybridization of genomic DNA (slot blot hybridization, reversed phase liquid hybridization or amplified mec A DNA).
Amplifikation von genomischer MRSA/MRSE DNAAmplification of genomic MRSA / MRSE DNA
Für die Amplifikation des mec A Gens oder Teilen davon wurden verschiedene Primer eingesetzt (z.B. EP 527 628 oder EP 526 876). In Verbindung mit den hier beschriebenen MRSA/MRSE spezifischen Oligo- und PolynuMeotidsonden sind die Primer 1 Sequenzprotokoll SEQ ID No 3 und Primer 2 Sequenzprotokoll SEQ ID No 4 besonders gut geeignet zur spezifischen Detektion von methicillin- resistenten Staphylococcen. Sie ergeben mit der genomischen DNA von methicillin- sensitiven Staphylococcen kein im Agarosegel sichtbares Amplifikationsprodukt. Genomische DNA von methiciUinresistenten Staphylococcen dagegen ergibt eine starke Amplifikationsbande, die gut mit der Oligonukleotidsonde oder der Poly- nuMeotidsonde hybridisiert und dadurch sehr gute Testsensitivitäten von < als 10 Keimen/ml Probenmaterial erreicht werden.Various primers were used for the amplification of the mec A gene or parts thereof (e.g. EP 527 628 or EP 526 876). In conjunction with the MRSA / MRSE-specific oligo and polynucleotide probes described here, the primer 1 sequence listing SEQ ID No 3 and primer 2 sequence listing SEQ ID No 4 are particularly well suited for the specific detection of methicillin-resistant staphylococci. With the genomic DNA of methicillin-sensitive staphylococci, they produce no amplification product visible in the agarose gel. Genomic DNA from methicin-resistant staphylococci, on the other hand, results in a strong amplification band that hybridizes well with the oligonucleotide probe or the polyMeotide probe, thereby achieving very good test sensitivities of <10 germs / ml sample material.
Bei der PCR-Amplifikation kann neben den 4 dNTPs (Desoxyadenosintriphosphat, Desoxyguanosintriphosphat, Desoxycytidintriphosphat und Thymidintriphosphat) zusätzlich z.B. Digoxigenin-dUTP in das Amplifikationsprodukt eingebaut werden. Dadurch kann das Amplifikationsprodukt mit einem Antidigoxigenin-Antikörper, der z.B. alk. Phosphatase gekoppelt enthält über einen Chemilumineszenztest mit AMPPD als Substrat oder einem Farbstofftest mit Brom-Chlor-Indolylphosphat und Nitroblautetrazolium ausgewertet werden.In the PCR amplification, in addition to the 4 dNTPs (deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate), e.g. Digoxigenin-dUTP can be incorporated into the amplification product. This allows the amplification product to be treated with an antidigoxigenin antibody, e.g. alk. Phosphatase coupled contains a chemiluminescence test with AMPPD as a substrate or a dye test with bromine-chloro-indolyl phosphate and nitro blue tetrazolium.
Alternativ besteht die Möglichkeit fluoreszenzmarkierte NuMeosidtriphosphate wie z.B. Fluoreszein-dUTP oder Cumarin-dUTPs in das Amplifikationsprodukt einzu- bauen und das Amplifikationsprodukt mit viel höherer Sensitivität als mit Ethidium- bromidanfärbung zu identifizieren.Alternatively, there is the possibility of fluorescence-labeled NuMeoside triphosphates such as Incorporate fluorescein dUTP or coumarin dUTPs into the amplification product and identify the amplification product with much higher sensitivity than with ethidium bromide staining.
Bei der Hairpin-Amplifikation (EP 427 074) wird z.B. ein T7/T3 Hairpinoligo- nuMeotid verwendet, das zusätzlich zur Hairpinsequenz eine Sequenz entsprechend den Oligonukleotidsequenzen aus der mec A Oligonukleotidsonde enthält (SEQ ID No 6-8). Mit T7/T3 Polymerase werden dann RNA-Transkripte hergestellt, die mit z.B. biotinylierten Capture-Gensonden (SEQ ID No 5) hybridisieren und an Streptavidin gecoateten magnetisierten Partikeln aus der Reaktionslösung separiert werden können. Der DNA/RNA Komplex kann dann durch DNA/RNA spezifische Antikörper (EP 339 686) detektiert werden, die mit alk. Phosphatase gekoppelt sind.For hairpin amplification (EP 427 074), for example, a T7 / T3 hairpin oligonucleotide is used which, in addition to the hairpin sequence, contains a sequence corresponding to the oligonucleotide sequences from the mec A oligonucleotide probe (SEQ ID No 6-8). RNA transcripts are then produced with T7 / T3 polymerase, which hybridize with, for example, biotinylated capture gene probes (SEQ ID No 5) and can be separated from the reaction solution with magnetized particles coated on streptavidin. The DNA / RNA complex can then be detected by DNA / RNA specific antibodies (EP 339 686), which with alk. Phosphatase are coupled.
Detektion von MRSA/MRSEDetection of MRSA / MRSE
Die MRSA/MRSE können direkt über ihre genomische DNA mit den in der Erfindung beschriebenen Gensonden nachgewiesen werden. Die Auswertung kann quantitativ über Slot Blot Hybridisierung oder Reversed Phase Flüssighybridisierung erfolgen. Allerdings ist die Testsensitivität mit ca. 105 Zellen/ml Probenmaterial relativ gering und damit für die Früherkennung von Infektionen mit diesen Erregern nur bedingt geeignet.The MRSA / MRSE can be detected directly via their genomic DNA using the gene probes described in the invention. The evaluation can be carried out quantitatively via slot blot hybridization or reversed phase liquid hybridization. However, the test sensitivity with approx. 10 5 cells / ml sample material is relatively low and therefore only of limited suitability for the early detection of infections with these pathogens.
Besser geeignet ist die Amplifikation des mec A Gens durch die in der Erfindung beschriebenen Primer, die für verschiedene Read Out Methoden mit Fluoreszenz, Biotin, Digoxigenin oder Enzyme wie alk. Phosphatase oder Horse Radish Peroxidase marMert werden können.The amplification of the mec A gene by the primers described in the invention, which are suitable for various read-out methods with fluorescence, biotin, digoxigenin or enzymes such as alk. Phosphatase or Horse Radish Peroxidase can be marbled.
Eine mögliche Read Out Methode ist die Anfarbung des durch Agarosegel- elektrophorese separierten Amplifikationsproduktes mit interkalierenden Agenzien wie Ethidiumbromid. Eine andere Möglichkeit ist der Einbau von fluoreszenz- mari ierten NuMeosidtriphosphaten in das DNA- oder RNA-Amplifikationsprodukt. Hierdurch wird eine deutliche Verbesserung der Testsensitivität erreicht.A possible read out method is the staining of the amplification product separated by agarose gel electrophoresis with intercalating agents such as ethidium bromide. Another possibility is the incorporation of fluorescence-mari ned NuMeosidtriphosphaten in the DNA or RNA amplification product. This leads to a significant improvement in test sensitivity.
Die zuverlässigste und sensitivste Methode, die gleichzeitig eine semiquantitative Auswertung ermöglicht ist die in der Erfindung beschriebene Methode der Hybridisierung der Amplifikationsprodukte mit den beschriebenen MRSA/MRSE spezifischen Gensonden. Diese Methode erlaubt darüber hinaus eine Quantifizierung der MRSA/MRSE in Minischem Probenmaterial (Figur 1). Beim Einbau von z.B. Digoxigenin-dUTP während der Amplifikation und Verwendung von biotinmariάerten Gensonden läßt sich der Hybridisierungskomplex an Streptavidin gecoateten magnetisierten Partikeln separieren und bei Verwendung von Antidigoxigenin- Antikörpern, die mit alk. Phosphatase gekoppelt sind, mit AMPPD als Substrat über Chemilumineszenz semi quantitativ auswerten. Beispiel 1The most reliable and most sensitive method, which at the same time enables semi-quantitative evaluation, is the method of hybridizing the amplification products with the MRSA / MRSE-specific gene probes described in the invention. This method also allows a quantification of the MRSA / MRSE in mini sample material (Figure 1). When, for example, digoxigenin-dUTP is incorporated during the amplification and use of biotin-marbled gene probes, the hybridization complex of streptavidin-coated magnetized particles can be separated and when using antidigoxigenin antibodies which are combined with alk. Phosphatase are coupled, with AMPPD as a substrate semi-quantitatively via chemiluminescence. example 1
Synthese von Oligonukleotidsonden und Starteroligonukleotiden (Primer)Synthesis of oligonucleotide probes and starter oligonucleotides (primers)
Die chemische Synthese der ausgewählten OligonuMeotidsonden und Starteroligo- nuMeotide (Primer) erfolgte nach der Phosphoramiditmethode von S.L. Beaucage und M. Caruthers, Tetrahedron Letters, 22, 1859, 1981. Folgende NuMeotidsequenzen wurden synthetisiert:The selected oligonucleotide probes and starter oligonucleotides (primers) were chemically synthesized using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981. The following NuMeotide sequences were synthesized:
OligonuMeotidsonde: SEQ ID No 1Oligonucleotide probe: SEQ ID No 1
PCR-Primer 1: SEQ ID No 3PCR primer 1: SEQ ID No 3
PCR-Primer 2: SEQ ID No 4 HAS-Capture-Probe, 5'-biotinyliert: SEQ ID No 5PCR primer 2: SEQ ID No 4 HAS capture probe, 5'-biotinylated: SEQ ID No 5
T3-Hairpinoligo mit mec A OligonuMeotidsequenz: SEQ ID No 6T3 hairpin oligo with mec A oligonucleotide sequence: SEQ ID No 6
T7-Hairpinoligo mit mec A OligonuMeotidsequenz: SEQ ID No 7T7 hairpin oligo with mec A oligonucleotide sequence: SEQ ID No 7
SP6-Hairpinoligo mit mec A OligonuMeotidsequenz: SEQ ID No 8SP6 hairpin oligo with mec A oligonucleotide sequence: SEQ ID No 8
PCR-Primer 3: SEQ ID No 9 PCR-Primer 4: SEQ ID No 10PCR primer 3: SEQ ID No 9 PCR primer 4: SEQ ID No 10
Die OligonuMeotidsonde wurde nach der Methode von Bollum, The enzymes Boyer ed, Vol 10, p 145, Academic Press New York, am 3' Ende mit Biotin-dUTP marMert. Die EndgruppenmarMerung wurde nicht radioaktiv mit Digoxigenin-dUTP vorgenommen (Chang, L.M.S., Bollum T.J., J. Biol. Chem. 246, 909, 1971).The oligonucleotide probe was marmered using the method of Bollum, The enzymes Boyer ed, Vol 10, p 145, Academic Press New York, at the 3 'end with Biotin-dUTP. End group marching was not radioactive with digoxigenin-dUTP (Chang, L.M.S., Bollum T.J., J. Biol. Chem. 246, 909, 1971).
In einem 50 μl Ansatz mit 10 μl Reaktionspuffer (Kaliumkakodylat 1 Mol/1; Tris/HCl 125 mmol/1; Rinderserumalbumin 1,25 mg/ml; pH 6,6; 25°C) 1-2 μg OligonuMeotid, 25 Einheiten Terminale Transferase, CoCl2 2,5 mmol/1 und 1 μl Biotin-d-UTP (1 mmol/1) werden nach 60 Minuten bei 37°C ca. 50 % 3'EndmarMe- rung erreicht.In a 50 μl batch with 10 μl reaction buffer (potassium cocodylate 1 mol / 1; Tris / HCl 125 mmol / 1; bovine serum albumin 1.25 mg / ml; pH 6.6; 25 ° C) 1-2 μg oligonucleotide, 25 units of terminals Transferase, CoCl 2 2.5 mmol / 1 and 1 μl biotin-d-UTP (1 mmol / 1) are reached after 60 minutes at 37 ° C approx. 50% 3'EndmarMering.
Beispiel 2Example 2
Konstruktion von Polynukleotidsonden für MRSA/MRSEConstruction of polynucleotide probes for MRSA / MRSE
Die PolynuMeotidsonde wurde durch PCR-Klonierung mit einem SureCloneTM Ligati on Kit der Firma Pharmacia Biochemicals erhalten. Für die PCR-Reaktion wurden 100 ng genomische DNA von MRSA/MRSE, 200 μmol dNTPs, 1,9 mM MgCl 2,2 μmol Primer 3 (SEQIDNO9) und Primer 4 (SEQEDNO10) und PCR-Puffer eingesetzt. Nach einer Initialschmelzung 5 Minuten bei 95°C wurde die DNA pro ZyMus 1 Minute bei 94°C denaturiert und dann 2 Minuten bei 50°C das Primerannealing durchgeführt. Anschließend wurde die Primerextension 2 Minuten bei 72°C durchgeführt nach 40 ZyMen wurde die Reaktion 20 Minuten bei 72°C behandelt und dann sofort die PCR-Klonierung durchgeführt. Dazu wurden die dATP Enden am 3' Ende des PCR-Produktes durch die 3'-5' ExonuMeaseal tivität des Klenows Fragmentes entfernt. Die PCR Fragmente wurden dann mit T4 PolynuMeotidMnase phosphoryliert und nach einer Phenol/Chloroform Behandlung und MicroSpin Säulenchromatographie das PCR-Produkt Blunt End ligiert in den mit boviner alkalischer Phosphatase entphosphorilierten pUC 18 VeMor. Anschließend erfolgte die Transformation und Selektion von PCR-Klonen nach Standardmethoden (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).The PolynuMeotidsonde was obtained by PCR cloning with a SureCloneTM Ligati on Kit from Pharmacia Biochemicals. For the PCR reaction 100 ng of genomic DNA from MRSA / MRSE, 200 μmol dNTPs, 1.9 mM MgCl 2.2 μmol primer 3 (SEQIDNO9) and primer 4 (SEQEDNO10) and PCR buffer were used. After an initial melting for 5 minutes at 95 ° C., the DNA per ZyMus was denatured for 1 minute at 94 ° C. and then the primer annealing was carried out for 2 minutes at 50 ° C. Subsequently, the primer extension was carried out at 72 ° C. for 2 minutes. After 40 cycles, the reaction was treated at 72 ° C. for 20 minutes and then the PCR cloning was carried out immediately. For this purpose, the dATP ends at the 3 'end of the PCR product were removed by the 3'-5' exonuase reactivity of the Klenows fragment. The PCR fragments were then phosphorylated with T4 PolynuMeotidMnase and after a phenol / chloroform treatment and MicroSpin column chromatography the PCR product Blunt End ligated into the pUC 18 VeMor dephosphorilized with bovine alkaline phosphatase. This was followed by the transformation and selection of PCR clones using standard methods (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).
Die aus den Polylmkersequenzen des VeMors isolierte 467 bp Sonde wurde durch random prime-Standardmethoden mit Biotin-d-UTP marMert. Alternativ wurde die Gensonde direkt durch PCR-Synthese mit Primer 3 und Primer 4 unter gleichzeitigem Einbau von 0,2 μmol Biotin-d-UTP hergestellt und in den Gentesten und Beispiel 6 eingesetzt.The 467 bp probe isolated from the polymer sequences of the VeMor was marmered by random prime standard methods with Biotin-d-UTP. Alternatively, the gene probe was produced directly by PCR synthesis with primer 3 and primer 4 with simultaneous incorporation of 0.2 μmol biotin-d-UTP and used in the genetic tests and example 6.
Beispiel 3Example 3
MRSA/MRSE spezifische DNA-Amplifikation mit der PCRMRSA / MRSE specific DNA amplification with the PCR
Die Amplifikation der Target-DNA wurde nach der Polymerase Chain Reaktion (EP 200 362; 201 184) durchgeführt.The amplification of the target DNA was carried out after the polymerase chain reaction (EP 200 362; 201 184).
Zur PCR-Reaktion wurden eingesetzt 1-1000 pg genomische DNA von methiciUinresistenten Staphylococcen, 2 μmol Primer 1 SEQ ID No 3 und Primer 2 SEQ ID No 4, 2,5 Units Taq-Polymerase von Cetus/PerMn-Elmer sowie jeweils 200 μmol dNTPS in einem Gesamtansatz von 100 μl PCR-Puffer (50 mM KC1, 10 mM Tris HCl pH 8,3, 1,5 mM MgCl2, und 0,01 % Gelatine. Die Amplifikation wurde in einem PCR-Prozessor der Firma Cetus/PerMn-Elmer durchgeführt. Für den Chemilumineszenztest nach Beispiel 6 wurde in der PCR zusätzlich 0,15 μmol Digoxigenin-d-UTP zur MarMerung des PCR-Produktes eingesetzt. Mit den Ansätzen wurde zunächst eine Initial Schmelzung der DNA 2 Minuten, 30 Sekunden bei 94°C durchgeführt, dann pro ZyMus 1 Minute bei 94°C die DNA denaturiert, 1 Minute 30 Sekunden bei 50°C das Primer-Annealing und 1 Minute 30 Sekunden bei 72°C die Primer-Extension durchgeführt. Nach 35 ZyMen wurde abschließend eine 10 Minuten-Extension bei 72°C durchgeführt und die Ansätze bei 4°C gekühlt.For the PCR reaction, 1-1000 pg of genomic DNA from methicin-resistant staphylococci, 2 μmol of primer 1 SEQ ID No 3 and primer 2 SEQ ID No 4, 2.5 units of Taq polymerase from Cetus / PerMn-Elmer and 200 μmol of dNTPS each were used in a total batch of 100 μl PCR buffer (50 mM KC1, 10 mM Tris HCl pH 8.3, 1.5 mM MgCl 2 , and 0.01% gelatin. The amplification was carried out in a PCR processor from Cetus / PerMn For the chemiluminescence test according to Example 6, an additional 0.15 μmol digoxigenin-d-UTP was used in the PCR to mar the PCR product. With the batches, the DNA was initially melted for 2 minutes, 30 seconds at 94 ° C, then the DNA was denatured for 1 minute at 94 ° C per cycle, the primer annealing for 1 minute 30 seconds at 50 ° C and 1 minute 30 The primer extension was carried out for seconds at 72 ° C. After 35 cycles, a 10 minute extension was then carried out at 72 ° C. and the batches were cooled at 4 ° C.
Beispiel 4Example 4
MRSA/MRSE spezifische RNA-Amplifikation mit HASMRSA / MRSE specific RNA amplification with HAS
Zur RNA-Amplifikation wurde eine Sandwich-Hybridisierung mit einer 5'biotinylierten Capture-Sonde (SEQ ID No 5) der genomischen DNA von Staphylococcen und dem 5'phosphorylierten HairpinoligonuMeotid SEQ ID No 6-8 durchgeführt wobei der Hybridisierungskomplex an Streptavidin gecoatete magnetisierbare Partikel gekoppelt war. Nach Ligierung von Capture Sonde und HairpinoligonuMeotid erfolgt die Transkriptionsamplifikation vom T7/T3 Hairpin mit Hilfe der entsprechenden RNA Polymerase.For RNA amplification, a sandwich hybridization was carried out using a 5'biotinylated capture probe (SEQ ID No 5) of the genomic DNA from staphylococci and the 5'phosphorylated hairpin oligonucleotide SEQ ID No 6-8, the hybridization complex being coupled to streptavidin-coated magnetizable particles was. After ligation of capture probe and hairpin oligonucleotide, the transcription amplification of the T7 / T3 hairpin is carried out using the corresponding RNA polymerase.
500 fmol Capture Oligo wurden mit 500 fmol Hairpinoligo und 10 bis 100 amol Target. -DNA in 50 μl T10E1 -Puffer 5 Minuten gekocht. 50 μl Target- Hybridisierungspuffer wurden hinzugegeben und dann 10 Minuten bei 52°C inkubiert. Dann wurden 100 μl Dynal Streptavidin Partikel zugegeben, 10 Minuten bei Raumtemperatur inkubiert und dann magnetisch separiert und der Überstand verworfen. Nach 3 x Waschen und Separieren mit Waschpuffer wurden 10 μl Ligase Premix-Puffer zugegeben und 15 Minuten bei 37°C ligiert. Nach 2 x Waschen und Separieren mit 200 μl Waschpuffer wurden 25 ml Transkriptionspuffer (IVT-MIX) mit T7-RNA-Polymerase zugegeben. Nach 2,5 Stunden bei 37°C war die RNA- Amplifikation beendet.500 fmol capture oligo were with 500 fmol hairpinoligo and 10 to 100 amol target. -DNA boiled in 50 μl T10E1 buffer for 5 minutes. 50 ul target hybridization buffer was added and then incubated at 52 ° C for 10 minutes. Then 100 μl of Dynal Streptavidin particles were added, incubated for 10 minutes at room temperature and then magnetically separated and the supernatant was discarded. After washing three times and separating with washing buffer, 10 μl of ligase premix buffer were added and ligated at 37 ° C. for 15 minutes. After washing twice and separating with 200 μl washing buffer, 25 ml transcription buffer (IVT-MIX) with T7 RNA polymerase were added. After 2.5 hours at 37 ° C the RNA amplification was finished.
TlOEl-Puffer lOmM Tris/HCl; pH 8, ImM EDTA Target 10 mlTlOEl buffer 10MM Tris / HCl; pH 8, ImM EDTA Target 10 ml
Hy bri di sierungspuff erHy bri diization puff er
20 x SSC 3 ml [6 X]20 x SSC 3 ml [6 X]
50 x Denhardt Lösung 0,2 ml [I X]50 x Denhardt's solution 0.2 ml [I X]
1 mg/ml Hefe tRNA 1 ml [100M μg/ml]1 mg / ml yeast tRNA 1 ml [100M μg / ml]
1 mg/ml Salmon sperm 1 ml [100 μg/ml] DNA (denaturiert)1 mg / ml Salmon sperm 1 ml [100 μg / ml] DNA (denatured)
Dextran sulphat l g [10 % w/v]Dextran sulphate l g [10% w / v]
Wasch-Puffer 100 mlWash buffer 100 ml
IM NaPhosphat, pH 7,2 10 ml [100 mM]IM NaPhosphate, pH 7.2 10 ml [100 mM]
10 % Tween-20 1 ml [0,1 %]10% Tween-20 1 ml [0.1%]
Ligase Premix 150 μlLigase premix 150 μl
5X Ligase Puffer (BRL) 30 μl [IX]5X Ligase Buffer (BRL) 30 μl [IX]
DEPC-Treated water 90 μlDEPC-Treated water 90 μl
1 U/μl Ligase (BRL) 30 μl [0,2 U/1] 1 U / μl ligase (BRL) 30 μl [0.2 U / 1]
IVT Premix 131 μl [Premix] [Final]IVT Premix 131 μl [Premix] [Final]
H2O 23 μlH 2 O 23 ul
3 mg/ml BSA 6,67 μl [152 ug/ml] [50 ug/ml]3 mg / ml BSA 6.67 μl [152 μg / ml] [50 μg / ml]
IM Tris, HC1, pH 8 16 μl [122 mM] [40 mM]IM Tris, HC1, pH 8 16 μl [122 mM] [40 mM]
IM MgCl 4 μl [30 mM] [10 mM]IM MgCl 4 μl [30 mM] [10 mM]
0,75 M DTT 5,33 μl [30 mM] [10 mM]0.75 M DTT 5.33 μl [30 mM] [10 mM]
IM NaCl 4 μl [30 mM] [10 mM]IM NaCl 4 μl [30 mM] [10 mM]
40 U/μl RNA-Guard 10 μl [3 U/μl] [1 U/μl]40 U / μl RNA-Guard 10 μl [3 U / μl] [1 U / μl]
0, IM ATP 2 μl [1,5 mM] [0,5 mM]0, IM ATP 2 μl [1.5 mM] [0.5 mM]
0,1M CTP 2 μl [1,5 mM] [0,5 mM]0.1M CTP 2 μl [1.5 mM] [0.5 mM]
0,1M UTP 2 μl [1,5 mM] [0,5 mM]0.1M UTP 2 μl [1.5 mM] [0.5 mM]
0,1m GTP 10 μl [7,5 mM] [2,5 mM]0.1m GTP 10 ul [7.5mM] [2.5mM]
70 U/μl T7 RNA 46 μl [24 U/μl] [8 U/μl] Polymerase70 U / ul T7 RNA 46 ul [24 U / ul] [8 U / ul] polymerase
Beispiel 5Example 5
Direkte Auswertung des AmplifikationsproduktesDirect evaluation of the amplification product
Zur direMen Auswertung des DNA-AmplifikationsproduMes wurden nach der Amplifikation interkalierende Agenzien wie z.B. Ethidiumbromid eingesetzt oder während der Amplifikation Fluoreszenz-NuMeotidtriphosphate wie z.B. Fluor- eszein dUTP oder Cumarin dUTP eingebaut. Es können auch Biotin-dUTP oder Digoxigenin-dUTP eingesetzt werden und mit Antikörper gekoppelter alk. Phos¬ phatase ein Farbstoff-Readout durchgeführt werden. Auch können bei geringerer Sensitivität entsprechend markierte Primer verwendet werden.For direct evaluation of the DNA amplification product, intercalating agents such as e.g. Ethidium bromide or during the amplification fluorescence NuMeotidtriphosphate such as e.g. Fluorescent dUTP or coumarin dUTP incorporated. Biotin-dUTP or digoxigenin-dUTP can also be used, and alk. Phosphatase a dye readout can be performed. Appropriately labeled primers can also be used if the sensitivity is lower.
Die bevorzugte Methode war der Einbau von Cumarin dUTP, weil dabei die beste Testsensitivität erreicht wurde. Das AmplifikationsproduM wurde auf ein 0,8 %iges Agarosegel aufgetragen und 30 Minuten bei 70 mA elektrophoretisiert. Die Fluoreszenz-Signale von methi- cillinsensitiven und methiciUinresistenten Staphylococcen wurden unter einem UV- Transilluminator direM ausgewertet.The preferred method was the incorporation of coumarin dUTP because the best test sensitivity was achieved. The amplification product was applied to a 0.8% agarose gel and electrophoresed at 70 mA for 30 minutes. The fluorescence signals from methicine-sensitive and methicin-resistant staphylococci were evaluated under a UV transilluminator direM.
Beispiel 6AExample 6A
Chemilumineszenz-Gensondentest von DNA-AmplifikationsproduktenChemiluminescence gene probe test of DNA amplification products
Durch die Kombination von geeigneten Target-Amplifikationsmethoden wie PCR (EP 200 362), LCR (EP 320 308), NASBA (EP 329 822), Qß (PCT 87/06270) oder HAS (EP 427 074) und der Gensondentechnik wird eine signifikante Sensitivitätssteigerung gegenüber den herkömmlichen Gensonden-Read Out Methoden erzielt.The combination of suitable target amplification methods such as PCR (EP 200 362), LCR (EP 320 308), NASBA (EP 329 822), Qß (PCT 87/06270) or HAS (EP 427 074) and the gene probe technique makes a significant Increased sensitivity compared to conventional gene probe read out methods.
Die Flüssighybridisierungsteste wurden als Reversed Phase Teste mit 100 ng 3'-biotinylierten OligonuMeotidsonde und amplifizierter DNA nach Beispiel 3 in einem Volumen von 50 μl durchgeführt.The liquid hybridization tests were carried out as reversed phase tests with 100 ng 3'-biotinylated oligonucleotide probe and amplified DNA according to Example 3 in a volume of 50 μl.
Nach 10-minütigem Erhitzen auf 100°C und anschließendem Abkühlen auf 0°C wurden 50 μl 2 x Boehringer Hybridisierungsmix zugegeben und 1 Stunde bei 45°C hybridisiert. Die magnetisi erbaren Beads wurden mit 1 x Boehringermix vorbehandelt und nach dem Separieren über einen Magneten die Flüssigkeit abpipettiert und der Hybridisierungsansatz zugegeben und 1/2 Stunde bei Raumtemperatur unter schwacher Bewegung inkubiert. Der gekoppelte Hybridisierungskomplex wurde mit den Beads separiert, die Restflüssigkeit abpipettiert und einmal mit Puffer A (2 x SSC; 0,1 % DS) bei Raumtemperatur, dann mit Puffer B (0,1 SSC; 0,1 % SDS) 2 x bei 45°C gewaschen.After heating to 100 ° C. for 10 minutes and then cooling to 0 ° C., 50 μl of 2 × Boehringer hybridization mix were added and the mixture was hybridized at 45 ° C. for 1 hour. The magnetizable beads were pretreated with 1 x Boehringer mix and, after separation using a magnet, the liquid was pipetted off and the hybridization mixture was added and incubated for 1/2 hour at room temperature with gentle agitation. The coupled hybridization complex was separated with the beads, the residual liquid was pipetted off and once with buffer A (2 x SSC; 0.1% DS) at room temperature, then with buffer B (0.1 SSC; 0.1% SDS) twice Washed 45 ° C.
Anschließend wurde die BlocMng Reaktion und Antikörper-Reaktion zum Nachweis der Hybridisierung über Chemilumineszenz durchgeführt. Die mit DNA beladenen Beads wurden 1 x mit 150 μl Waschpuffer (0,1 M Maleinsäure, 0,1 MNaCl pH 7,5, 0,3 % Tween 20) behandelt und nach dem Separieren und Abpipettieren des Waschpuffers 400 μl Puffer 2 (0,1M Maleinsäure; 0,15M NaCl, pH 7,5; 1 % BlocMng Reagenz (Boehringer)) zugegeben. Nach 1/2 Stunde Inkubation bei Raumtemperatur wurde separiert, abpipettiert und 100 μl Antikörperkonjugat-Lösung (AK 1:10000 in Puffer 2) zugegeben und 1/2 Stunde bei Raumtemperatur inkubiert, dann separiert, abpipettiert und mit 400 μl Waschpuffer behandelt 2 x 15 Minuten bei schwacher Bewegung. Anschließend wurde separiert und mit 150 μl Puffer 3 (0,1M Tris/HCl Puffer mit 0,1M NaCl und 50 mM MgCl2 pH 9,5) behandelt. Es wurde wieder separiert und mit 100 μlDeteMionslösung mit AMPPD 1 : 100 in Puffer 3 15 Minuten bei 37°C im Wasserbad inkubiert, dann die Chemilumineszenz im Lumineszenz-Photometer bei 477 nm (Lumacounter von Lumac) gemessen.The BlocMng reaction and antibody reaction for detection of hybridization via chemiluminescence was then carried out. The beads loaded with DNA were treated 1 × with 150 μl washing buffer (0.1 M maleic acid, 0.1 MNaCl pH 7.5, 0.3% Tween 20) and, after separating and pipetting off the washing buffer, 400 μl buffer 2 (0 , 1M maleic acid; 0.15M NaCl, pH 7.5; 1% BlocMng reagent (Boehringer)) added. After incubation for 1/2 hour at room temperature, the mixture was separated, pipetted off and 100 μl of antibody conjugate solution (AK 1: 10000 in buffer 2) added and incubated for 1/2 hour at room temperature, then separated, pipetted off and treated with 400 μl washing buffer 2 × 15 minutes with gentle agitation. It was then separated and treated with 150 μl buffer 3 (0.1M Tris / HCl buffer with 0.1M NaCl and 50 mM MgCl 2 pH 9.5). It was separated again and incubated with 100 μl of detergent solution with AMPPD 1: 100 in buffer 3 for 15 minutes at 37 ° C. in a water bath, then the chemiluminescence was measured in the luminescence photometer at 477 nm (Lumacounter from Lumac).
Mit diesem Test können DNA-Mengen entsprechend 106 bis 101 Staphylococcus Keime detektiert werden. Die gleiche Detektionsgrenze von 10 Keimen pro ml Blut wurde auch nach Zugabe von unspezifischer Blut-DNA erreicht. Blut-DNA alleine gibt nur geringe Background-Signale im Hybridisierungstest.With this test, DNA amounts corresponding to 10 6 to 10 1 Staphylococcus germs can be detected. The same detection limit of 10 germs per ml of blood was reached even after the addition of non-specific blood DNA. Blood DNA alone gives only little background signals in the hybridization test.
Beispiel 6 BExample 6 B
Chemilumineszenz-SchnelltestRapid chemiluminescence test
In Beispiel 6 B wird ein vereinfachtes alternatives Chemilumi-neszenz-Testverfahren zu Beispiel 6 A beschrieben das bei gleicher Testsensitivität in 1 Stunde durchfuhrbar ist und automatisiert werden kann.Example 6 B describes a simplified alternative chemiluminescence test method to Example 6 A which can be carried out in 1 hour with the same test sensitivity and can be automated.
Die Flüssighybridisierungsteste wurden als Reversed Phase Teste mit lOOng 3'- biotinylierter Gensonde und amplifizierter DNA nach Beispiel 3 in einem Volumen von 50μl durchgeführt.The liquid hybridization tests were carried out as reversed phase tests with 100 ng 3'-biotinylated gene probe and amplified DNA according to Example 3 in a volume of 50 μl.
Nach 10 minütigem Erhitzen auf 100° C und anschließendem Abkühlen auf 0° C wurden 50 μl 2x Hybridisierungsmix (50ml 20XS S C, l g Blocking Reagenz(Boehringer),2 ml 10% iges Lauroylsarcosin,200μl 20 %iges SDS ad 100 ml bidest H2O) zugegeben und 5- 10 Minuten bei 37° C hybridisiert (OligonuMeotidsonde). Die magnetischen Beads wurden mit lx Hybridisiermix vorbehandelt und nach dem Separieren über einen Magneten die Flüssigkeit ab¬ pipettiert, der Hybridisierungsansatz und lOOμl lx Hybridsiermix zugegeben und 5- 10 Minuten bei Raumtemperatur unter schwacher Bewegung inkubiert.Der gekop¬ pelte Hybridisierungskomplex wurde mit den Beads separiert,die Restflüssigkeit abpipettiert und einmal mit Puffer B(0,1 SSC;0,1%SDS) lx gewaschen. Anschließend wurde die BlocMng Reaktion und Antikörper-ReaMion zum Nachweis der Hybridisierung über Chemilumineszenz durchgeführtDie mit DNA beladenen Beads wurden lx mit 500μl Puffer 2 (0,1M Maleinsäure;0,15M NaCl pH7,5;l% Bloc ng Reagenz (Boehringer)) zugegeben.Nach 5 Minuten Inkubation bei Raumtemperatur wurde separiert,abpipettiert und 250μl Aritikörperkonjugat-Lösung (AK 1:2500 in Puffer 2) zugeben und 10 Minuten bei Raumtemperatur inkubiert, dann separiert, abpipettiert und mit 500μl Waschpuffer behandelt 2x 30 Sekunden, lx 2 Minuten bei schwacher Bewegung.Es wurde dann mit lOOμl Detektionslösung mit AMPPD 1:100 in Puffer 3 10 Minuten bei 37° C im Wasserbad inkubiert, dann die Chemilumineszenz im Lumineszenz-Photometer bei 477 nm (Lumacounter von Lumac) gemessen.After heating for 10 minutes at 100 ° C and then cooling to 0 ° C, 50 μl 2x hybridization mix (50 ml 20XS SC, Ig blocking reagent (Boehringer), 2 ml 10% lauroylsarcosine, 200 μl 20% SDS ad 100 ml bidest H 2 O) added and hybridized at 37 ° C. for 5-10 minutes (oligonucleotide probe). The magnetic beads were pretreated with 1 × hybridization mix and, after separation using a magnet, the liquid was pipetted off, the hybridization mixture and 100 μl 1 × hybridization mix were added and incubated for 5-10 minutes at room temperature with gentle agitation. The coupled hybridization complex was separated with the beads , the remaining liquid is pipetted off and washed once with buffer B (0.1 SSC; 0.1% SDS) once. The BlocMng reaction and antibody reaction were then carried out to detect the hybridization via chemiluminescence. The beads loaded with DNA were added 1x with 500μl buffer 2 (0.1M maleic acid; 0.15M NaCl pH7.5; 1% Bloc ng reagent (Boehringer)) After 5 minutes of incubation at room temperature, the mixture was separated, pipetted off and 250μl of antibody conjugate solution (AK 1: 2500 in buffer 2) were added and incubated for 10 minutes at room temperature, then separated, pipetted off and treated with 500μl wash buffer 2x 30 seconds, lx 2 minutes The movement was then incubated with 100 μl detection solution with AMPPD 1: 100 in buffer 3 for 10 minutes at 37 ° C. in a water bath, then the chemiluminescence was measured in the luminescence photometer at 477 nm (Lumacounter from Lumac).
Mit diesem Test können DNA-Mengen entsprechend 106 bis 101 Staphylococcus Keime detektiert werden.Die gleiche Detektionsgrenze von 10 Keimen pro ml Blut wurde auch nach Zugabe von unspezifischer Blut-DNA erreicht.Blut-DNA alleine gibt nur geringe Background-Signale im Hybridisierungstest.With this test DNA amounts corresponding to 10 6 to 10 1 Staphylococcus germs can be detected. The same detection limit of 10 germs per ml blood was reached even after the addition of non-specific blood DNA. Blood DNA alone gives only little background signals in the hybridization test .
Beispiel 7Example 7
Chemilumineszenz-Gensondentest von RNA-AmplifikationsproduktenChemiluminescence gene probe test of RNA amplification products
Die MRSA/MRSE spezifischen RNA-Transskripte (ca. 0,5 pmol) wurden mit dem Capture Oligo (4 pmol) und 10 μl 3 x Transkriptionspuffer 2 in einem Gesamtvolumen von 30 μl hybridisiert. Dazu wurde der Ansatz 2 Minuten bei 98°C denaturiert, dann 10 Minuten bei 57°C hybridisiert und dann 70 μl H2O hinzugefügt. Nach Zugabe von 100 μl Dynal Streptavidin Partikel wurde 10 Minuten bei Raumtemperatur inkubiert, magnetisch separiert und der Überstand verworfen. Dann wurde 3 x mit 200 μl Waschpuffer gewaschen und separiert. Anschließend wurde 2 x mit 200 μl Antikörper-Bindepuffer gewaschen. Es wurden 250 μl Anti-DNA/RNA-Antikörper (0,05 μg/ml) gekoppelt mit alk. Phosphatase zugegeben und 15 Minuten bei 37°C unter Schütteln inkubiert. Dann wurde 3 x mit 200 μl -Antikörper-Bindepuffer gewaschen. Die magnetisierbaren Partikel wurden in 200 μl Antikörper-Bindepuffer resuspendiert und in frischen Teströhrchen mit 0,5 ml AMPPD-Lösung 20 Minuten bei 37°C inkubiert. Die Auswertung erfolgte wie im Beispiel 6 in einem Luminometer. 3 x Transcriptionspuffer 10 ml [3 X] [Final]The MRSA / MRSE-specific RNA transcripts (approx. 0.5 pmol) were hybridized with the capture oligo (4 pmol) and 10 μl 3 × transcription buffer 2 in a total volume of 30 μl. For this purpose, the mixture was denatured for 2 minutes at 98 ° C., then hybridized for 10 minutes at 57 ° C. and then 70 μl H 2 O were added. After adding 100 μl of Dynal Streptavidin particles, the mixture was incubated at room temperature for 10 minutes, magnetically separated and the supernatant was discarded. Then it was washed 3 times with 200 μl washing buffer and separated. The mixture was then washed twice with 200 μl of antibody binding buffer. 250 μl anti-DNA / RNA antibodies (0.05 μg / ml) were coupled with alk. Phosphatase added and incubated for 15 minutes at 37 ° C with shaking. Then was washed 3 times with 200 ul antibody binding buffer. The magnetizable particles were resuspended in 200 μl of antibody binding buffer and incubated in fresh test tubes with 0.5 ml of AMPPD solution at 37 ° C. for 20 minutes. The evaluation was carried out as in Example 6 in a luminometer. 3 x transcription buffer 10 ml [3 X] [Final]
20 X SSC 3 ml [6 X] [2 x]20 X SSC 3 ml [6 X] [2 x]
IM HEPES, pH 7,4 0,3 ml [30 mM] [10 mM]IM HEPES, pH 7.4 0.3 ml [30 mM] [10 mM]
0,5 mM EDTA, pH 8,0 60 μl [3 mM] [1 mM]0.5 mM EDTA, pH 8.0 60 μl [3 mM] [1 mM]
10 % SDS 0,75 ml [0,75 % w/v] [0,25 % w/v]10% SDS 0.75 ml [0.75% w / v] [0.25% w / v]
Waschpuffer 100 mlWash buffer 100 ml
IM NaPhosphat, pH 7,2 10 ml [100 mM]IM NaPhosphate, pH 7.2 10 ml [100 mM]
10 % Tween-20 1 ml [0,1 %]10% Tween-20 1 ml [0.1%]
b Bindungspuffer [0,1M Tris.HCl, 0,1M NaCl, 0,1 % BSA, 0,1 % Tween]b Binding buffer [0.1M Tris.HCl, 0.1M NaCl, 0.1% BSA, 0.1% Tween]
Anti DNA/RNA:AlkPhos 5 mlAnti DNA / RNA: AlkPhos 5 ml
21,6 ug/ml Antikörper 11,7 μl [0,05 μg/ml]21.6 µg / ml antibody 11.7 µl [0.05 µg / ml]
Ab Bindungspuffer 4,99 mlFrom binding buffer 4.99 ml
Substrat/Enhancer (Tropix, Boston, MA) 10 mlSubstrate / enhancer (Tropix, Boston, MA) 10 ml
AMPPD Substrat 0,17 mlAMPPD substrate 0.17 ml
Saphire Enhancer 1 mlSapphire Enhancer 1 ml
Diethylamine Substrat Puffer, pH 9,5 8,83 ml Beispiel 8Diethylamine substrate buffer, pH 9.5 8.83 ml Example 8
Isolierung von Staphylococcen-NukleinsäureIsolation of staphylococcal nucleic acid
Das mit Staphylococcen infizierte Probenmaterial, z.B. infiziertes Blut, wurde bei 8000 UpM 5 bis 10 Minuten zentrifugiert, der Überstand abpipettiert und verworfen. Das Sediment (ca. 180 bis 200 μl) wurde mit 50 μl TE mit 15 %iger Saccharose versetzt, 20 μl Lysozym + Lysostaphin (10+ 5 mg/ml in bidest.H2O) zugegeben und 15 Minuten bei 37°C inkubiert. Die weitere Aufarbeitung erfolgte mit einem Quiamp-DNA-Kit der Firma Diagen. Nach Zugabe von 25 μl Proteinase K und 235 μl AL Puffer wurde gemischt und 10 Minuten bei 70°C behandelt. Dann in Eis abgekühlt und 240 μl 100 %iges Ethanol zugegeben und nach kurzem Mischen auf die Qiagen-Säule aufgetragen. Anschließend wurde die Säule 1 Minute bei 8000 UpM zentrifigiert, das Eluat verworfen. Es wurde 700 μl Wasch-Puffer aufgetragen und nochmals 1 Minute bei 8000 UpM zentrifugiert. Anschließend wurde nochmals mit der gleichen Menge AW Puffer gewaschen und 1 Minute bei 8000 UpM und 10 Sekunden bei 14000 UpM zentrifugiert und das Eluat verworfen. Es wurden dann 200 μl bidest H2O auf die Säule aufgetragen und 1 Minute bei 8000 UpM zentrifugiert. Das Eluat enthält die für die Amplifikation einsetzbare gereinigte NuMeinsäurelösung.The sample material infected with staphylococci, for example infected blood, was centrifuged at 8000 rpm for 5 to 10 minutes, the supernatant was pipetted off and discarded. The sediment (approx. 180 to 200 μl) was mixed with 50 μl TE with 15% sucrose, 20 μl lysozyme + lysostaphin (10+ 5 mg / ml in bidist. H 2 O) was added and incubated for 15 minutes at 37 ° C . Further processing was carried out using a Diagen Quiamp DNA kit. After addition of 25 μl Proteinase K and 235 μl AL buffer, the mixture was mixed and treated at 70 ° C. for 10 minutes. Then cooled in ice and 240 μl of 100% ethanol were added and, after brief mixing, applied to the Qiagen column. The column was then centrifuged for 1 minute at 8000 rpm, the eluate was discarded. 700 μl of washing buffer was applied and centrifuged again at 8000 rpm for 1 minute. The mixture was then washed again with the same amount of AW buffer and centrifuged for 1 minute at 8000 rpm and 10 seconds at 14000 rpm and the eluate was discarded. 200 μl bidest H 2 O were then applied to the column and centrifuged at 8000 rpm for 1 minute. The eluate contains the purified nucleic acid solution which can be used for the amplification.
Beispiel 9Example 9
Nachweis von MRSA/MRSE in klinischem ProbenmaterialDetection of MRSA / MRSE in clinical specimens
Die MRSA/MRSE DNA wurde aus dem Minischen Probenmaterial nach der in Beispiel 8 beschriebenen Methode isoliert. Das Staphylococcen-DNA-Lysat wurde dann mit Hilfe geeigneter Amplifikationsmethoden wie im Beispiel 5 beschrieben mit MRSA/MRSE spezifischen OligonuMeotid-Primern amplifiziert. Die amplifizierte NuMeinsäure wurde dann mit der OligonuMeotidsonde SEQ ID No 1 oder der PolynuMeotidsonde SEQ ID No 2 hybridisiert und der unter stringenten Bedingungen sich ausbildende spezifische Hybridisierungskomplex aus amplifizierter Staphylococcen-NuMeinsäure und Gensonden-DNA wurde mit magnetisierbaren Partikeln der Firma Dynal separiert und wie im Beispiel 6 durch Chemilumineszenz- Read Out quantitativ bestimmt. In den mit MRSA/MRSE infizierten Blutlysaten wurden mec A Gen-spezifische Hybridisierungssignale noch in einer Konzentration von 101 Keimen problemlos nachgewiesen.The MRSA / MRSE DNA was isolated from the mini sample material according to the method described in Example 8. The staphylococcal DNA lysate was then amplified using suitable amplification methods as described in Example 5 with MRSA / MRSE-specific oligonucleotide primers. The amplified nucleic acid was then hybridized with the oligonucleotide probe SEQ ID No 1 or the polynucleotide probe SEQ ID No 2 and the specific hybridization complex of amplified staphylococcal nucleic acid and gene probe DNA which developed under stringent conditions was separated with magnetizable particles from Dynal and as in the example 6 quantified by chemiluminescence read out. In the blood lysates infected with MRSA / MRSE, mec A gene-specific hybridization signals were still easily detected in a concentration of 10 1 germs.
Beispiel 10 - Quantitativer Nachweis von MRSA/MRSE MRSA/MRSE-Nukleinsäure wurden wie im Beispiel 8 aus klinischem Proben¬ material, z.B. Blut, isoliert. Die Amplifikation wurde wie in Beispiel 3 beschrieben durchgeführt. Die Zyklenzahl wurde auf 25 Zyklen beschränkt um eine gute Korrelation zwischen Zellzahl und Chemilumineszenzsignal in dem klinisch rele¬ vanten Bereich von 103 bis 10 Keimen zu erreichen. Neben den klinischen Proben wurden Proben mit MRSA MRSE DNA entsprechend den Zellzahlen von 106 bis 10° Zellen in 500 ng Blut-DNA parallel amplifiziert und im Chemilumineszenztest analysiert (Beispiel 6). Die Figur 1 zeigt die Chemilumineszenzsignale von 106 bis 10 MRSA MRSE im Blut im Vergleich zu parallel amplifizierten Testproben mit 103 und 102 Keimen, die mikrobiologisch nachgewiesen worden waren. Das Diagramm zeigt, daß die Chemilumineszenzsignale aus dem Zell¬ zahl-DNA-Standard (103-102) sehr gut mit den Chemilumineszenzsignalen der Testproben mit 103 und 102 Keimen vergleichen lassen und der Chemilumin¬ eszenztest zur Quantifizierung der MRSA/MRSE z.B. im Blut eingesetzt werden kann.Example 10 - Quantitative detection of MRSA / MRSE MRSA / MRSE nucleic acid were isolated from clinical sample material, for example blood, as in Example 8. The amplification was carried out as described in Example 3. The number of cycles was limited to 25 cycles in order to achieve a good correlation between the number of cells and the chemiluminescence signal in the clinically relevant range from 10 3 to 10 germs. In addition to the clinical samples, samples with MRSA MRSE DNA corresponding to the cell numbers from 10 6 to 10 ° cells were amplified in parallel in 500 ng blood DNA and analyzed in the chemiluminescence test (Example 6). FIG. 1 shows the chemiluminescence signals of 10 6 to 10 MRSA MRSE in the blood in comparison to test samples with 10 3 and 10 2 germs amplified in parallel, which had been detected microbiologically. The diagram shows that the chemiluminescence signals from the cell number DNA standard (10 3 -10 2 ) can be compared very well with the chemiluminescence signals of the test samples with 10 3 and 10 2 germs and the chemiluminescence test for quantifying the MRSA / MRSE eg can be used in the blood.
Abbildung 1illustration 1
In Abbildung 1 ist ein Chemilumineszenztest zum semiquantitativen Nachweis von MRSA dargestellt. Die Amplifikation wurde wie im Beispiel 3 durchgeführt,die Zyklenzahl dabei auf 25 Zyklen beschränkt um eine gute Korrelation zwischen Zellzahl und Chemilumineszenzsignal in dem klinisch relevanten Bereich von 1000 bis 10 Keimen /ml Probenmaterial zu erreichen.Figure 1 shows a chemiluminescence test for the semi-quantitative detection of MRSA. The amplification was carried out as in Example 3, the number of cycles being limited to 25 cycles in order to achieve a good correlation between cell number and chemiluminescence signal in the clinically relevant range from 1000 to 10 germs / ml of sample material.
Das Diagramm zeigt die Chemilumineszenzsignale eines Zellstandards von 106 bis 10 MRSA in Blut im Vergleich zu parallel mitamplifizierten Testproben mit 1000 und 100 Keimen, die mikrobiologisch nachgewiesen worden waren. Aus dem Diagramm ist ersichtlich, daß die Chemilumineszenzsignale von dem Zell-DNA-Standard(102-103) sich sehr gut mit den Chemilumineszenzsignalen der Testproben mit 1000 und 100 Keimen vergleichen lassen und der Chemilumin¬ eszenztest zur semiquantitativen Bestimmung von MRSA z.B. in Blut eingesetzt werden kann. SEQUENZPROTOKOLLThe diagram shows the chemiluminescence signals of a cell standard of 10 6 to 10 MRSA in blood in comparison to test samples with 1000 and 100 germs, which were also amplified in parallel, and which had been detected microbiologically. From the diagram it can be seen that the chemiluminescence signals from the cell DNA standard (10 2 -10 3 ) can be compared very well with the chemiluminescence signals of the test samples with 1000 and 100 germs and the chemiluminescence test for the semi-quantitative determination of MRSA, for example in Blood can be used. SEQUENCE LOG
(1) ALGEMEINE INFORMATION:(1) GENERAL INFORMATION:
(i) ANMELDER:(i) APPLICANT:
(A) NAME: Bayer AG (B) STRASSE: Bayerwerk(A) NAME: Bayer AG (B) ROAD: Bayerwerk
(C) ORT: Leverkusen(C) LOCATION: Leverkusen
(E) LAND: Deutschland(E) COUNTRY: Germany
(F) POSTLEITZAHL: 51368(F) POSTAL NUMBER: 51368
(G) TELEPHON: 0214/3061455 (H) TELEFAX: 0214/303482(G) TELEPHONE: 0214/3061455 (H) TELEFAX: 0214/303482
(ii) ANMELDETITEL: Spezifische Gensonden und Verfahren zum quantitativen Nachweis von methicillin-resistenten Staphylococcen(ii) REGISTRATION TITLE: Specific gene probes and methods for the quantitative detection of methicillin-resistant staphylococci
(iii) ANZAHL DER SEQUENZEN: 10(iii) NUMBER OF SEQUENCES: 10
(iv) COMPUTER-LESBARE FORM:(iv) COMPUTER READABLE FORM:
(A) DATENTRÄGER: Floppy disk(A) DISK: Floppy disk
(B) COMPUTER: IBM PC co patible(B) COMPUTER: IBM PC co patible
(C) BETRIEBSSYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25(C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentin Release # 1.0, Version # 1.25
(EPA)(EPA)
(2) INFORMATION ZU SEQ ID NO: 1:(2) INFORMATION ABOUT SEQ ID NO: 1:
(i) SEQUENZ CHARAKTERISTIKA: (A) LÄNGE: 35 Basenpaare (B) ART: N kleinsäure(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: N small acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch) (iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus(A) ORGANISM: Staphylococcus aureus
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 1: TATGTATGGC ATGAGTAACG AAGAATATAA TAAAT 35(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TATGTATGGC ATGAGTAACG AAGAATATAA TAAAT 35
(2) INFORMATION ZU SEQ ID NO: 2:(2) INFORMATION ABOUT SEQ ID NO: 2:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 467 Basenpaare (B) ART: Nukleinsäure(A) LENGTH: 467 base pairs (B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus(A) ORGANISM: Staphylococcus aureus
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 2:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ATGATTATGG CTCAGGTACT GCTATCCACC CTCAAACAGG TGAATTATTA GCACTTGTAA 60 GCACACCTTC ATATGACGTC TATCCATTTA TGTATGGCAT GAGTAACGAA GAATATAATA 120ATGATTATGG CTCAGGTACT GCTATCCACC CTCAAACAGG TGAATTATTA GCACTTGTAA 60 GCACACCTTC ATATGACGTC TATCCATTTA TGTATGGCAT GAGTAACGAA GAATATAATA 120
AATTAACCGA AGATAAAAAA GAACCTCTGC TCAACAAGTT CCAGATTACA ACTTCACCAG 180AATTAACCGA AGATAAAAAA GAACCTCTGC TCAACAAGTT CCAGATTACA ACTTCACCAG 180
GTTCAACTCA AAAAATATTA ACAGCAATGA TTGGGTTAAA TAACAAAACA TTAGACGATA 240GTTCAACTCA AAAAATATTA ACAGCAATGA TTGGGTTAAA TAACAAAACA TTAGACGATA 240
AAACAAGTTA TAAAATCGAT GGTAAAGGTT GGCAAAAAGA TAAATCTTGG GGTGGTTACA 300AAACAAGTTA TAAAATCGAT GGTAAAGGTT GGCAAAAAGA TAAATCTTGG GGTGGTTACA 300
ACGTTACAAG ATATGAAGTG GTAAATGGTA ATATCGACTT AAAACAAGCA ATAGAATCAT 360 CAGATAACAT TTTCTTTGCT AGAGTAGCAC TCGAATTAGG CAGTAAGAAA TTTGAAAAAG 420ACGTTACAAG ATATGAAGTG GTAAATGGTA ATATCGACTT AAAACAAGCA ATAGAATCAT 360 CAGATAACAT TTTCTTTGCT AGAGTAGCAC TCGAATTAGG CAGTAAGAAA TTTGAAAAAG 420
GCATGAAAAA ACTAGGTGTT GGTGAAGATA TACCAAGTGA TTATCCA 467GCATGAAAAA ACTAGGTGTT GGTGAAGATA TACCAAGTGA TTATCCA 467
(2) INFORMATION ZU SEQ ID NO: 3:(2) INFORMATION ON SEQ ID NO: 3:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 18 Basenpaare (B) ART: Nukleinsäure(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
( ii ) ART DES MOLEKÜLS : DNS (genomisch) ( iii ) HYPOTHETISCH : NEIN (iii) ANTISENSE: NEIN(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus(A) ORGANISM: Staphylococcus aureus
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 3: TAAAGATGAT GCAGTTAT 18(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TAAAGATGAT GCAGTTAT 18
(2) INFORMATION ZU SEQ ID NO: 4:(2) INFORMATION ABOUT SEQ ID NO: 4:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 24 Basenpaare(A) LENGTH: 24 base pairs
(B) ART: Nukleinsäure (C) STRANGFORM: Einzel(B) TYPE: nucleic acid (C) STRAND FORM: single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETISCH: NEIN(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NEIN (vi) URSPRÜNLICHE HERKUNFT:(iii) ANTISENSE: NO (vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus(A) ORGANISM: Staphylococcus aureus
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4: TGGATAATCA CTTGGTATAT CTTC 24(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TGGATAATCA CTTGGTATAT CTTC 24
(2) INFORMATION ZU SEQ ID NO: 5: (i) SEQUENZ CHARAKTERISTIKA:(2) INFORMATION ABOUT SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 26 Basenpaare(A) LENGTH: 26 base pairs
(B) ART: Nukleinsäure(B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear (ii) ART DES MOLEKÜLS: DNS (genomisch)(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETISCH: NEIN(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NEIN(iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus (xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 5: TCCATTTATG TATGGCATGA GTAACG 26(A) ORGANISM: Staphylococcus aureus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCCATTTATG TATGGCATGA GTAACG 26
(2) INFORMATION ZU SEQ ID NO: 6:(2) INFORMATION ON SEQ ID NO: 6:
(i) SEQUENZ CHARAKTERISTIKA: (A) LÄNGE: 66 Basenpaare(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs
(B) ART: Nukleinsäure(B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch) (iii) HYPOTHETISCH: NEIN(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NEIN(iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus/ T3 Phage(A) ORGANISM: Staphylococcus aureus / T3 Phage
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 6 : AAGAATATAA TAAATTAACC GTCCCTTTAG TGAGGGTAAA TTTTTTATTT ACCCTCACTA 60(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAGAATATAA TAAATTAACC GTCCCTTTAG TGAGGGTAAA TTTTTTATTT ACCCTCACTA 60
AAGGGA 66 (2) INFORMATION ZU SEQ ID NO: 7:AAGGGA 66 (2) INFORMATION ABOUT SEQ ID NO: 7:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 62 Basenpaare (B) ART: Nukleinsäure(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus/ T7 Phage(A) ORGANISM: Staphylococcus aureus / T7 phage
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 7: AAGAATATAA TAAATTAACC GCTATAGTGA GTCGTATTAT TTTTTAATAC GACTCACTAT 60(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AAGAATATAA TAAATTAACC GCTATAGTGA GTCGTATTAT TTTTTAATAC GACTCACTAT 60
AG S2AG S2
(2) INFORMATION ZU SEQ ID NO: 8:(2) INFORMATION ABOUT SEQ ID NO: 8:
(i) SEQUENZ CHARAKTERISTIKA: (A) LÄNGE: 74 Basenpaare(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 base pairs
(B) ART: Nukleinsäure(B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch) (iii) HYPOTHETISCH: NEIN(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NEIN(iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus/ SP6 Phage(A) ORGANISM: Staphylococcus aureus / SP6 phage
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 8: AAGAATATAA TAAATTAACC GCTATACTGT CACCTAAATC GTATGTTTTT CATACGATTT 60 AGGTGACACT ATAG 74(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AAGAATATAA TAAATTAACC GCTATACTGT CACCTAAATC GTATGTTTTT CATACGATTT 60 AGGTGACACT ATAG 74
(2) INFORMATION ZU SEQ ID NO: 9:(2) INFORMATION ON SEQ ID NO: 9:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 18 Basenpaare (B) ART: Nukleinsäure(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS : Staphylococcus aureus(A) ORGANISM: Staphylococcus aureus
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 9:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATGATTATGG CTCAGGTA 1 (2) INFORMATION ZU SEQ ID NO: 10:ATGATTATGG CTCAGGTA 1 (2) INFORMATION ABOUT SEQ ID NO: 10:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 19 Basenpaare(A) LENGTH: 19 base pairs
(B) ART: Nukleinsäure (C) STRANGFORM: Einzel(B) TYPE: nucleic acid (C) STRAND FORM: single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch)(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETISCH: NEIN(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NEIN (vi) URSPRÜNLICHE HERKUNFT:(iii) ANTISENSE: NO (vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Staphylococcus aureus(A) ORGANISM: Staphylococcus aureus
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 10: TATCTTCACC AACACCTAG 19 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TATCTTCACC AACACCTAG 19

Claims

Patentansprüche claims
1. Eine Oligonukleotidsonde, die mit DNA oder RNA von methiciUinresistenten Staphylococcen hybridisiert, dadurch gekennzeichnet, daß sie die Nukleotidsequenz wie im Sequenzprotokoll SEQ ID No 1 enthält oder mutierte Sequenzen davon oder markierte Sequenzen davon.1. An oligonucleotide probe that hybridizes with DNA or RNA of methicin-resistant staphylococci, characterized in that it contains the nucleotide sequence as in the sequence listing SEQ ID No 1 or mutated sequences thereof or labeled sequences thereof.
2. Eine Polynukleotidsonde, die mit DNA oder RNA von methiciUinresistenten Staphylococcen hybridisiert, dadurch gekennzeichnet, daß sie 467 Basen oder Teile davon enthält und die Nukleotidsequenz wie im Sequenzprotokoll SEQ ID No 2 oder Teile davon enthält oder mutierte Sequenzen davon oder markierte Sequenzen davon.2. A polynucleotide probe which hybridizes with DNA or RNA of methicin-resistant staphylococci, characterized in that it contains 467 bases or parts thereof and contains the nucleotide sequence as in the sequence listing SEQ ID No 2 or parts thereof or mutated sequences thereof or labeled sequences thereof.
3. Starteroligonukleotide (Primer) für die Amplifikation und den spezifischen Nachweis von Teilen des mec A Gens, dadurch gekennzeichnet, daß sie die Nukleotidsequenz wie im Sequenzprotokoll SEQ ID No 3 und 4 enthalten.3. Starter oligonucleotides (primers) for the amplification and the specific detection of parts of the mec A gene, characterized in that they contain the nucleotide sequence as in the sequence listing SEQ ID No 3 and 4.
4. Hairpinoligonukleotide für die RNA-Amplifikation und den spezifischen Nachweis von Teilen des mec A Gens, dadurch gekennzeichnet, daß sie die4. Hairpin oligonucleotides for the RNA amplification and the specific detection of parts of the mec A gene, characterized in that they
Nukleotidsequenz wie im Sequenzprotokoll SEQ ID No 6 bis 8 enthalten.Nucleotide sequence as contained in the sequence listing SEQ ID No 6 to 8.
5. Verfahren zum Nachweis von methiciUinresistenten Staphylococcen in Probenmaterial, dadurch gekennzeichnet, daß5. A method for the detection of methicin resistant staphylococci in sample material, characterized in that
a) Oligonukleotid- oder Polynukleotidsonden nach Anspruch 1 und 2 zur spezifischen Hybridisierung und Detektion von methicillinresistentera) oligonucleotide or polynucleotide probes according to claim 1 and 2 for specific hybridization and detection of methicillin resistant
Nukleinsäure von Staphylococcen eingesetzt werdenNucleic acid from staphylococci can be used
b) Oligo- und Polynukleotidsonden mit Hilfe von bekannten Methoden mit Reportermolekülen markiert werdenb) Oligo and polynucleotide probes are labeled with reporter molecules using known methods
c) die Nukleinsäure von methicillinresistenten/methicillinsensitiven Staphylococcen direkt oder bevorzugt nach Amplifikation mit an sich bekannten Verfahren zur Reaktion mit den Sonden eingesetzt wird oder direkt ohne weitere Hybridisierung analysiert wird d) der Hybridisierungskomplex über die Markierung der amplifϊzierten Target-DNA ein direktes Maß für den Nachweis und die Menge an methiciUinresistenten Staphylococcen liefert.c) the nucleic acid of methicillin-resistant / methicillin-sensitive staphylococci is used directly or preferably after amplification using methods known per se for reaction with the probes or is analyzed directly without further hybridization d) the hybridization complex provides a direct measure of the detection and the amount of methicin-resistant staphylococci via the labeling of the amplified target DNA.
6. Ein Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß Oligo- nukleotidsonden nach Anspruch 1 eingesetzt werden.6. A method according to claim 5, characterized in that oligonucleotide probes according to claim 1 are used.
7. Ein Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß eine Poly¬ nukleotidsonde nach Anspruch 2 verwendet wird.7. A method according to claim 5, characterized in that a poly nucleotide probe according to claim 2 is used.
8. Ein Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß für die Amplifikation von Teilen des mec A Gens die Primer nach Anspruch 3 eingesetzt werden.8. A method according to claim 5, characterized in that the primers according to claim 3 are used for the amplification of parts of the mec A gene.
9. Ein Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß für die Amplifikation von Teilen des mec A Gens Hairpinoligonukleotide nach Anspruch 4 eingesetzt werden.9. A method according to claim 5, characterized in that hairpin oligonucleotides according to claim 4 are used for the amplification of parts of the mec A gene.
10. Testkit, enthaltend eine oder mehrere der Oligonukleotide nach Ansprüchen 1 bis 4 sowie Puffer und andere Lösungen zur praktischen Durchführung einer Analyse auf Methicillinresistenz in klinischen Probenmaterial. 10. Test kit containing one or more of the oligonucleotides according to claims 1 to 4 as well as buffers and other solutions for the practical implementation of an analysis for methicillin resistance in clinical sample material.
PCT/EP1994/003553 1993-11-08 1994-10-28 Specific gene probes and methods for quantitative detection of methicillin-resistant staphylococci WO1995013395A1 (en)

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