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WO1995013395A1 - Analyseurs specifiques de genes et procedes de determination quantitative de staphylocoques resistant a la methicilline - Google Patents

Analyseurs specifiques de genes et procedes de determination quantitative de staphylocoques resistant a la methicilline 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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Wolfgang Springer
Rainer Endermann
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Bayer AG
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Bayer AG
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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

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Abstract

L'invention concerne de nouveaux analyseurs de gènes, ainsi que des procédés de détermination quantitative de staphylocoques résistant à la méthicilline.
PCT/EP1994/003553 1993-11-08 1994-10-28 Analyseurs specifiques de genes et procedes de determination quantitative de staphylocoques resistant a la methicilline Ceased WO1995013395A1 (fr)

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Application Number Priority Date Filing Date Title
DEP4338119.7 1993-11-08
DE4338119A DE4338119A1 (de) 1993-11-08 1993-11-08 Spezifische Gensonden und Verfahren zum quantitativen Nachweis von methicillinresistenten Staphylococcen

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WO1995013395A1 true WO1995013395A1 (fr) 1995-05-18

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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1160333A3 (fr) * 2000-05-29 2004-01-07 Tosoh Corporation Oligonucléotides et méthodes pour la détection du gène mecA du Staphylococcus aureus résistant à la méthicilline
WO2006097233A3 (fr) * 2005-03-18 2006-11-16 Eppendorf Ag Determination de la resistance aux antibiotiques du staphylocoque dore
US7838221B2 (en) 2005-10-11 2010-11-23 Geneohm Sciences, Inc. Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA)
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
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