DE4338119A1 - Specific gene probes and methods for the quantitative detection of methicillin-resistant staphylococci - Google Patents

Description

The proportion of methicillin-resistant strains has increased since the mid-1970s with staphylococcal infections from a few percent up to 70%. While the appearance of these strains initially only at large university clinics was restricted, today these resistant strains are increasingly found in Primary care hospitals (Paul et al., Abstr. 23 ICAAC (1991)).

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. B. Kathe to go out.

Methicillin-resistant Staphylococcus are of particular clinical importance aureus (MRSA) and Staphylococcus epidermidis (MRSE) during Staphylococcus hemolytic infections occur only occasionally.

MRSA / MRSE are against all β-lactam antibiotics, such as penicillins, cephalospo rine, peneme and carbapeneme resistant. 80% of the MRSA / MRSE strains there is also resistance to other classes of antibiotics such as macrolides and amino glycosides and quinolones.

It is therefore particularly important to treat the effect as early as possible initiate seed antibiotic. The alternatives for the treatment of infections with methicillin-resistant staphylococci are limited to a few antibiotics. Vankomycin is the antibiotic of choice. Another glycopeptide antibiotic,  Teicoplanin, has only recently been introduced. Another therapy option is the combination of antibiotics such as ciprofloxacin with rifampicin.

The classic diagnosis of MRSA / MRSE is time-consuming and labor-intensive demands the in vitro cultivation of the strains on plates with subsequent phy siological identification with the help of test systems such as B. "api staph" (BioM´rieux) and determination of antibiotic resistance by serial dilution tests (MIC values) or agar diffusion tests (inhibitory tests).

In contrast to those in sensitive, methicillin-resistant staphylococci Strains of existing penicillin binding protein genes an additional so-called mec A gene coding for the penicillin binding protein PBP2a. This has PBP2a an extremely low affinity for all β-lactam antibiotics and therefore enables as transpeptidase, cell wall synthesis in the presence of β-lactam concentrate ions that inhibit all other PBPs (Neu, Science 257, 1064 (1992)).

Because this mec A gene is present in all methicillin-resistant staphylococci and the homology between the different mec A genes is very high, on the basis of this gene, a rapid DNA diagnostics with speci fish gene probes combined with known DNA or RNA application methods driving to develop.

The present invention describes specific oligonucleotide and polynucleo tide probes and their use for the rapid detection of methicillin resistance staphylococci directly in the clinical specimen.

• 1. The gene probes were prepared 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
  • b) does not occur in other penicillin binding protein genes and
  • c) all methicillin-resistant staphylococci with MIC values between 4 to 120 µg / ml clearly recorded.
• 2. The genomic DNA of methicillin resistant staphylococci was by methods known per se, which have been adapted for Staphylococcus, isolated.
• 3. The application of parts of the mec A gene was through specific Primer from the coding and non-coding strand of the mec A gene with simultaneous marking of the amplification product mi Digoxi genin-dUTP using known amplification methods, preferably the PCR-DNA amplification method (EP 200 362) or the HAS-RNA-Am plication method (EP 427 074) carried out.
• 4. The specific amplification product was detected by Hy bridization of the amplification product with the above, biotiny gene probes. This means that with a detection of 10 germs achieved significantly higher sensitivity than by direct detection of the Amplification product in the gel, with a maximum of 10³ germs be shown.
• 5. The hybridization complex is coupled with streptavidin magneti separated particles.
• 6. The evaluation of the hybridization complex formed as a measure of the Amount or presence of methicillin resistant staphylococci is carried out by a chemiluminescence test with antidigoxigenin antibodies those with alk. Phosphatase coupled (PCR) or by DNA / RNA Antibodies with alk. Phosphatase are coupled (HAS amplification).

The evaluation can also be amplified when using an external Staphylococcal DNA standard semi-quantitative to determine the amount of methicillin-resistant staphylococci used in clinical specimens become.  

Gene probe diagnostics especially in connection with amplification technology ken is a quick, specific and highly sensitive method that used to be detection of the pathogen at the DNA / RNA level. The technology can be direct without in vitro cultivation in the test material. she is based on the DNA / RNA hybridization technique, d. H. the specific in vitro Binding of complementary single-stranded nucleic acid to form Watson-Crick base pairs. The DNA / DNA or DNA / RNA double formed strands are also known as DNA hybrids. To detect the specific DNA or RNA of a pathogen by the hybridization reaction are com complementary sequence-specific gene probes are used. These gene probes have been removed neither short, chemically synthesized oligonucleotide probes with a length of 10 to 50 nucleotides or DNA / RNA fragments of 0.5 to 10 kb, which by recombinant genetic engineering were produced.

The gene probes can be photochemically (N.Dattagupta, et al., Biochem. 177, 85, 1989) or enzymatically by nick translation (Rigby, P.W.J. et al., J. Mol. Biol. 113, 237, 1977) or random primed techniques (Feinberg and Vogelstein, Anal. Biochem. 132, 6, 1983) with a radioactive or non-radioactive label be provided. Markings with 32 P NTPs or not are suitable for this radioactive labels with digoxigenin-dUTP, biotin-dUTP or direct Labeling with enzymes such as alk. Phosphatase or Horseradish Peroxidase.

For the specific hybridization between the nucleic acid to be detected of the pathogen and the pathogen-specific gene probe become the nucleic acids next by denaturation (heat or alkali treatment) in single strands separates and then under stringent conditions by temperature, ionic strength the buffer and organic solvents are achieved, specifically with each other other hybridizes. The gene binds under suitable hybridization conditions probe only on complementary sequences of the DNA or RNA to be detected. This hybridization reaction can be carried out in various test formats e.g. B. as a festival phase hybridization to a carrier such as e.g. B. Nitrocellulose Coupled Target DNA or gene probe or as a liquid hybridization. The Evaluation (Read Out) is carried out by marking the gene probe with a Reporter molecule as listed above or as reversed as shown here Phase hybridization system over the target DNA during amplification  is labeled with digoxigenin-dUTP and the gene probe that binds to magnetic particles are marked with biotin. The hybridization complex from Target DNA and labeled gene probe is removed after removing non-hybri quantified DNA using the reporter molecule used. This Read out can be done directly with fluorescence labeling or radioactive Labeling or indirectly using enzyme tests and immunological methods Antibody conjugates, the enzymes such as the alk. Contain phosphatase and then enable a color reaction or chemiluminescence reaction.

The test sensitivity with this gene probe diagnosis is in the range from 10⁵ to 10⁶ germs based on the detection of single genes. An increase in the test sensitivity can be combined with DNA or RNA amplification techniques such as 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 can multiply the DNA to be detected by a factor of 10 be achieved. By combining amplification and hybridization this enables the detection of individual DNA molecules.

Since 10-1-10³ germs / ml count in infections in the blood, offers with this technology the possibility of early detection of methicillin resistant infection courses.

In the present invention, new specific and particularly sensitive Gene probes for methicillin-resistant staphylococci are described.

The new gene probes were developed from gene areas of the mec A gene that specific and particularly strong hybridization signals for methicillin resistant Have staphylococci and both methicillin resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), but no signal methicillin sensitive staphylococci. The construction of oligo nucleotide probes and polynucleotide probes for methicillin-resistant staphylococci is described in the invention. GenBank and EMBL nucleotide sequence Databases were not homologous 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.

There will also be a procedure for using these gene probes in Hybri described in which a specific hybridization with Target DNA of methicillin-resistant staphylococci is carried out.

Furthermore, in this invention the combination of these hybridizations method using amplification techniques, especially hairpin amplification method (EP 427 074), described by which a very strong improvement in Test sensitivity is achieved.

There has also been the use of these gene probes and test procedures for detection of methicillin-resistant staphylococci in clinical specimens ben. Another great advantage of this method is the possibility of one semi-quantitative determination of the amount of methicillin-resistant infections men in the sample material.

Selection and synthesis of oligonucleotide probes

For the selection of suitable specific oligonucleotide probes, the Nucleotide sequence of the mec A gene from Staphylococcus aureus and Staphylococcus epidermidis (Ryffel et al., Gene 94, 137 (1990) Song et al., FEBS Letters 221, 167 (1987). Sequences that were conserved in the mec A gene from MRSA / MRSE occur, but no homology to the have known PBPs. The preferred oligonucleotide probe is in the Sequence listing SEQ ID No 1 described. With this oligonucleotide probe can perform solid phase hybridization tests with e.g. B. digoxigenin end-labeled Probes or liquid hybridization tests with e.g. B. Photodigoxigenin labeled genomic DNA and biotin-labeled oligonucleotide probes with which the Hybridization complex with the help of streptavidin coupled magnetic Particles are separated, performed. The disadvantage of this method is the relatively low detection limit of 10⁵ pathogens. Because in the early stages of Infection, far lower concentrations of pathogens occur, this ver do not go out for an early diagnosis.  

In the present invention, therefore, the described gene probe technique combined with DNA and RNA amplification methods such as B. the PCR technique (EP 200 362, EP 201 184) and hairpin amplification technique (EP 427 074).

The process in which the mec A gene or parts thereof are first derived from the genomic DNA are amplified and then amplification product hybridized specifically with the oligonucleotide probe compared to the pure amplification diagnostics the following advantages:

• 1. The amplification products often contain depending on the Process conditions such as annealing temperature, primer and enzyme concentration, primer sequence and MgCL2 concentration and used Polymerase, by non-specific by-products by primer mismatching when staining the amplification products in the gel or with fluorescence Mark false positive results during amplification reflect.
• 2. In addition, there is the significantly lower test sensitivity with direct evaluation tion of the PCR products, which is 10³ germs, while in combination with gene probe hybridization and a chemiluminescence read out a detection of 10 germs per ml of sample material is possible without any problems.

The chemical synthesis of the selected oligonucleotide probe was carried out according to the S.L.'s phosphoramidite method Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981.

Construction of the polynucleotide probe

The polynucleotide probe was PCR cloned with a SureCloneTM Ligation kit from Pharmacia Biochemicals. Was received cloned into the pUC18 vector, a polynucleotide probe of 467 nucleotides the nucleotide sequence described in the sequence listing SEQ ID No 2.  

The 467 Sp probe was removed from the vector by restriction enzyme digestion, Aga Rose gel electrophoresis and electroelution isolated and then by standard markie methods (random prime, 3'end group marking) with z. B. Biotin-d UTP marked. Alternatively, the probe was directly by PCR synthesis with primer 3 and 4 produced 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 tion or amplified mec A DNA) can be used.

Amplification of genomic MRSA / MRSE DNA

Various were used for the amplification of the mec A gene or parts thereof Primer used (e.g. EP 527 628 or EP 526 876). In connection with this MRSA / MRSE-specific oligo and polynucleotide probes described the primer 1 sequence listing SEQ ID No 3 and primer 2 sequence listing SEQ ID No 4 particularly well suited for the specific detection of methicillin resistant staphylococci. They result from methicil's genomic DNA lens-sensitive staphylococci no amplification product visible in the agarose gel. Genomic DNA from methicillin-resistant staphylococci, on the other hand, gives one strong amplification band that works well with the oligonucleotide probe or the poly hybridizes nucleotide probe and therefore very good test sensitivities of <than 10 Germination / ml sample material can be reached.

In addition to the 4 dNTPs (deoxyadenosine triphosphate, Deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate) additionally z. B. digoxogenin dUTP in the amplification product the. This allows the amplification product to be treated with an antidigoxigenin anti body, the z. B. alk. Phosphatase coupled contains via a chemiluminescence test with AMPPD as substrate or a dye test with bromine-chloro-indo lylphosphate and nitro blue tetrazolium are evaluated.

Alternatively there is the possibility of fluorescence-labeled nucleoside triphosphates such as e.g. B. fluorescein dUTP or coumarin dUTPs in the amplification product  build and the amplification product with much higher sensitivity than with Identify ethidium bromide staining.

In hairpin amplification (EP 427 074) z. B. a T7 / T3 Hairpinoligo nucleotide used, in addition to the hairpin sequence a sequence corresponding contains the oligonucleotide sequences from the mec A oligonucleotide probe (SEQ ID No 6-8). RNA transcripts are then produced using T7 / T3 polymerase posed with z. B. biotinylated capture gene probes (SEQ ID No 5) hybridi sieren and on streptavidin coated magnetic particles from the reaction solution can be separated. The DNA / RNA complex can then pass through DNA / RNA specific antibodies (EP 339 686) can be detected, which with alk. Phosphatase are coupled.

Detection of MRSA / MRSE

The MRSA / MRSE can directly with their genomic DNA with those in the Gene probes described invention can be detected. The evaluation can quantitative via slot blot hybridization or reversed phase liquid hybridization tion. However, the test sensitivity is around 10⁵ cells / ml Sample material relatively small and therefore for the early detection of infections only partially suitable with these pathogens.

The amplification of the mec A gene by the in the invention is more suitable described primers which are suitable for different read-out methods with fluorescence, Boitin, digoxigenin or enzymes such as alk. Phosphatase or Horse Radish Peroxidase can be labeled.

A possible read out method is the staining of the agarose gel electrophoresis separated amplification product with intercalating agents like ethidium bromide. Another possibility is the incorporation of fluorescence labeled nucleoside triphosphates in the DNA or RNA amplification product. This leads to a significant improvement in test sensitivity.

The 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 quantification of the MRSA / MRSE in clinical sample material ( Fig. 1). When installing z. B. Digoxigenin-dUTP during the amplification and use of biotin-labeled gene probes, the hybridization complex of streptavidin-coated magnetic particles can be separated and, when using antidi goxigenin antibodies, which with alk. Phosphatase are coupled, using AMIPPD as a substrate to evaluate semiquantitatively via chemiluminescence.

example 1 Synthesis of oligonucleotide probes and starter oligonucleotides (primers)

The chemical synthesis of the selected oligonucleotide probes and starter oligo nucleotides (primers) were made using the phosphoramidite method of S.L. Beaucage and M. Caruthers, Tetrahedron Letters, 22, 1859, 1981. The following nucleotide sequences were synthesized:

Oligonucleotide probe: SEQ ID No 1
PCR primer 1: SEQ ID No 3
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 designed according to the method of Bollum, The enzymes Boyer ed, Vol 10, p 145, Academic Press New York, at the 3 ′ end with Biotin-dUTP marked. The end group labeling was not radioactive with digoxigenin dUTP (Chang, L.M.S., Bollum T.J., J. Biol. Chem. 246, 909, 1971).

In a 50 µl mixture with 10 µl reaction buffer (potassium cocodylate 1 mol / l; Tris / HCl 125 mmol / l; Bovine serum albumin 1.25 mg / ml; pH 6.6; 25 ° C) 1-2 µg Oligonucleotide, 25 units of terminal transferase, CoCl₂ 2.5 mmol / l and 1 µl Biotin-d-UTP (1 mmol / l) becomes about 50% 3′Endmar after 60 minutes at 37 ° C achieved.

Example 2 Construction of polynucleotide probes for MRSA / MRSE

The polynucleotide probe was PCR cloned with a SureCloneTM Obtained ligation 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 (SEQIDNO10) and PCR Buffer used. After an initial melting at 95 ° C. for 5 minutes, the DNA per cycle for 1 minute at 94 ° C and then for 2 minutes at 50 ° C Primerannealing performed. Then the primer extension was 2 minutes carried out at 72 ° C after 40 cycles, the reaction was at 72 ° C for 20 minutes treated and then immediately performed the PCR cloning. For this, the dATP ends at the 3'end of the PCR product due to the 3'-5'exonuclease activity of the Klenows fragment removed. The PCR fragments were then labeled with T4 Polynucleotide kinase phosphorylated and after phenol / chloroform treatment and MicroSpin column chromatography ligated the PCR product into the blunt end pUC 18 vector dephosphorilized with bovine alkaline phosphatase. The transformation and selection of PCR clones then followed Standard Methods (Maniatis et al., Molecular Cloning) Cold Spring Harbor Laboratory Press, 1989).

The 467 SP probe isolated from the polylinker sequences of the vector was analyzed by random prime standard methods labeled with biotin-d-UTP. Alternatively, the Gene probe directly by PCR synthesis with primer 3 and primer 4 below Simultaneous installation of 0.2 µmol Biotin-d-UTP manufactured and in the Genetic testing and Example 6 used.

Example 3 MRSA / MRSE specific DNA amplification with the PCR

The amplification of the target DNA was done after the polymerase chain reaction (EP 200 362; 201 184).

1-1000 pg of genomic DNA from were used for the PCR reaction methicillin resistant staphylococci, 2 µmol primer 1 SEQ ID No 3 and Primer 2 SEQ ID No 4, 2.5 units Taq polymerase from Cetus / Perkin-Elmer and each 200 µmol dNTPS in a total of 100 µl PCR buffer (50 mM KCl, 10 mM Tris / HCl pH 8.3, 1.5 mM MgCl₂, and 0.01% gelatin. The ampli Specification was carried out in a PCR processor from Cetus / Perkin-Elmer leads. For the chemiluminescence test according to Example 6, PCR was also carried out 0.15 µmol digoxidgonin-d-UTP was used to label the PCR product.  

Initially, the batches were used to initially melt the DNA for 2 minutes, 30 Performed at 94 ° C for seconds, then DNA at 94 ° C for 1 minute per cycle denatured, 1 minute 30 seconds at 50 ° C the primer annealing and 1 minute The primer extension was carried out for 30 seconds at 72 ° C. After 35 cycles finally a 10 minute extension at 72 ° C and the approaches chilled at 4 ° C.

Example 4 MRSA / MRSE specific RNA amplification with HAS

For hybrid amplification, a sandwich hybridization with a 5′biotinylated capture probe (SEQ ID No 5) of the genomic DNA from Staphiococcen and the 5′phosphorylated hairpin oligonucleotide SEQ ID No 6-8 carried out, the hybridization complex coated on streptavidin magnetic particle was coupled. After alloy of capture probe and Hairpin oligonucleotide, the transcription amplification is carried out by the T7 / T3 hairpin with the help of the corresponding RNA polymerase.

500 fmol capture oligo were mixed with 500 fmol hairpinoligo and 10 to 100 amol Target.-DNA boiled in 50 µl T10E1 buffer for 5 minutes. 50 µl target Hybridization buffers were added and then 10 minutes at 52 ° C incubated. Then 100 ul Dynal Streptavidin particles were added, 10 minutes incubated at room temperature and then magnetically separated and the supernatant discarded. After 3 × washing and separation with washing buffer, 10 ul Ligase premix buffer added and ligated for 15 minutes at 37 ° C. To 2 × washing and separation with 200 μl washing buffer were 25 ml Transcription buffer (IVT-MIX) with T7 RNA polymerase added. After 2.5 RNA amplification was complete for hours at 37 ° C.

T10E1 buffer 10 mM Tris / HCl; pH 8, 1mM EDTA

Example 5 Direct evaluation of the amplification product

For the direct evaluation of the DNA amplification product after the Amplification intercalating agents such as B. Ethidium bromide or fluorescence nucleotide triphosphates such as e.g. B. fluorine eszein dTUP or coumarin dUTP built in. It can also use biotin or dUTP Digoxigenin-dUTP are used and alk. Phos phatase a dye readout. Can also with less Sensitivity appropriately labeled primers can be used.

The preferred method was the incorporation of coumarin dUTP because it was 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 methi cillin sensitive and methicillin resistant staphylococci were exposed to UV Transilluminator evaluated directly.

Example 6 Chemiluminescence gene probe test of DNA amplification products

By combining 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 gene probe technology becomes significant Increased sensitivity compared to the conventional gene probe read out Methods achieved.

The liquid hybridization tests were performed as reversed phase tests with 100 ng 3'-biotinylated oligonucleotide probe and amplified DNA according to Example 3 in a volume of 50 ul carried out.

After heating to 100 ° C for 10 minutes and then cooling to 0 ° C 50 ul 2 × Boehringer hybridization mix were added and 1 hour at Hybridized at 45 ° C. The magnetic beads were mixed with 1 × Boehringer mix pretreated and after separating the liquid with a magnet pipetted off and the hybridization batch added and 1/2 hour at Incubated room temperature with gentle agitation. The coupled hybridisie The complex was separated with the beads, the remaining liquid was pipetted off and once with buffer A (2 × SSC; 0.1% DS) at room temperature, then with buffer B (0.1 SSC; 0.1% SDS) washed 2 × at 45 ° C.

The blocking reaction and antibody reaction then became Detection of hybridization carried out via chemiluminescence. The one with DNA loaded beads were washed 1 × with 150 µl washing buffer (0.1 M maleic acid, 0.1 MNaCl pH 7.5, 0.3% Tween 20) treated and after the separation and Pipette off the washing buffer 400 µl buffer 2 (0.1 M maleic acid; 0.15 M NaCl, pH 7.5; 1% blocking reagent (Boehringer)) added. After 1/2 hour Incubation at room temperature was separated, pipetted off and 100 µl  Antibody conjugate solution (AK 1: 10000 in buffer 2) added and 1/2 hour incubated at room temperature, then separated, pipetted off and washed with 400 µl buffer treated 2 × 15 minutes with weak movement. Then was separated and with 150 µl buffer 3 (0.1 M Tris / HCl buffer with 0.1 M NaCl and 50 mM MgCl₂ pH 9.5) treated. It was separated again and with detection solution with AMPPD 1: 100 in buffer 3 for 15 minutes at 37 ° C in a water bath incubated, then the chemiluminescence in the luminescence photometer at 477 nm (Lumacounter from Lumac) measured.

With this test DNA amounts corresponding to 106 to 101 Staphylococcus Germs can be detected. The same detection limit of 10 germs per ml Blood was also obtained after the addition of non-specific blood DNA. Blood DNA alone gives only little background signals in the hybridization test.

Example 7 Chemiluminescence gene probe test of RNA amplification products

The MRSA / MRSE-specific RNA transcripts (approx. 0.5 pmol) were generated with the Capture oligo (4 pmol) and 10 µl 3 × transcription buffer 2 in one Total volume of 30 ul hybridized. The batch was added for 2 minutes 98 ° C denatured, then hybridized at 57 ° C for 10 minutes and then 70 ul H₂O added. After adding 100 µl of Dynal Streptavidin particles, 10 Incubated for minutes at room temperature, magnetically separated and the supernatant discarded. The mixture was then washed 3 × with 200 μl washing buffer and separated. The mixture was then washed twice with 200 μl of antibody binding buffer. There were 250 µl anti-DNA / RNA antibody (0.05 µg / ml) coupled with alk. Phosphatase added and incubated for 15 minutes at 37 ° C. with shaking. Then 3 × washed with 200 ul antibody binding buffer. The magnetic particles were resuspended in 200 µl antibody binding buffer and in fresh test tubes Incubate 0.5 ml AMPPD solution for 20 minutes at 37 ° C. The evaluation was carried out as in example 6 in a luminometer.

From binding buffer [0.1 M Tris.HCl, 0.1 M NaCl, 0.1% BSA, 0.1% Tween]

Example 8 Isolation of staphylococcal nucleic acid

The sample material infected with staphylococci, e.g. B. infected blood was in Centrifuged at 8000 rpm for 5 to 10 minutes, the supernatant was pipetted off and discarded. The sediment (approx. 180 to 200 µl) was treated with 50 µl TE with 15%  Sucrose added, 20 µl lysozyme + lysostaphin (10+ 5 mg / ml in bidist. H₂O) added and incubated for 15 minutes at 37 ° C. The rest of the work was done with a Quiamp DNA kit from Diagen. After adding 25 µl Proteinase K and 235 ul AL buffer was mixed and 10 minutes at 70 ° C treated. Then cooled in ice and 240 ul 100% ethanol added and applied to the Qiagen column after brief mixing. Then the Centrifuged column at 8000 rpm for 1 minute, the eluate was discarded. It became 700 µl Wash buffer applied and centrifuged again at 8000 rpm for 1 minute. It 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 that Eluate discarded. Then 200 ul bidest H₂O were applied to the column and centrifuged at 8000 rpm for 1 minute. The eluate contains those for Purified nucleic acid solution that can be used for amplification.

Example 9 Detection of MRSA / MRSE in clinical specimens

The MRSA / MRSE DNA was extracted from the clinical specimen according to the Example 8 isolated method. The Staphylccoccen DNA lysate was then using suitable amplification methods as described in Example 5 amplified with MRSA / MRSE specific oligonucleotide primers. The amplified nucleic acid was then with the oligonucleotide probe SEQ ID No 1 or the polynucleotide probe SEQ ID No 2 hybridizes and the under stringent Conditions developing specific hybridization complex amplified staphylococcal nucleic acid and gene probe DNA was analyzed with magnetic particles from Dynal separated and as in Example 6 by Chemiluminescence Read Out quantified.

In the blood lysates infected with MRSA / MRSE, mec A became gene-specific Hybridization signals still in a concentration of 10 ′ germs without problems proven.  

Example 10 Quantitative detection of MRSA / MRSE

MRSA / MRSE nucleic acid were as in Example 8 from clinical sample material, eg. Blood isolated. The amplification was carried out as described in Example 3. The number of cycles was limited to 25 cyclones in order to achieve a good correlation between cell number and 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 10⁶ to 100 lines in 500 ng blood DNA were amplified in parallel and analyzed in the chemiluminescence test (Example 6). Fig. 1 shows the chemiluminescence signals from 10⁶ to 10 MRSA / MRSE in the blood in comparison to test samples amplified in parallel with 10³ and 10² germs, which had been detected microbiologically. The diagram shows that the chemiluminescence signals from the cell number DNA standard (10³-10²) compare very well with the chemiluminescence signals of the test samples with 10³ and 10² germs and the chemiluminescence test to quantify the MRSA / MRSE z. B. can be used in the blood.

Sequence listing

• (1) GENERAL INFORMATION:
  • (i) APPLICANT:
    • (A) NAME: Bayer AG
    • (B) ROAD: Bayerwerk
    • (C) LOCATION: Leverkusen
    • (E) COUNTRY: Germany
    • (F) POSTAL NUMBER: 51368
    • (G) TELEPHONE: 0214/3061455
    • (H) TELEFAX: 0214/303482
  • (ii) REGISTRATION TITLE: Specific gene probes and methods for the quantitative detection of methicillin-resistant staphylococci
  • (iii) NUMBER OF SEQUENCES: 10
  • (iv) COMPUTER READABLE FORM:
    • (A) DISK: Floppy disk
    • (B) COMPUTER: IBM PC compatible
    • (C) OPERATING SYSTEM: PC-DOS / MS-DOS
    • (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.25 (EPA)
• (2) INFORMATION ABOUT SEQ ID NO: 1:
  • (1) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 35 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
• (2) INFORMATION ABOUT SEQ ID NO: 2:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 467 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
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  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
• (2) INFORMATION ON SEQ ID NO: 3:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 18 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
• (2) INFORMATION ABOUT SEQ ID NO: 4:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 24 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
  • (xi) SEQUENCE DESCRIPTION: SEQ ID No: 4:
• (2) INFORMATION ON SEQ ID NO: 5:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 26 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
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• (2) INFORMATION ON SEQ ID NO: 6:
  • (i) SEQUENCE CHARACTERISTICS:
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    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
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  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus / T3 Phage
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
• (2) INFORMATION ON SEQ ID No: 7:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 62 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus / T7 phage
  • (xi) SEQUENCE DESCRIPTION: SEQ ID No: 7:
• (2) INFORMATION ABOUT SEQ ID NO: 8:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 74 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus / SP6 phage
  • (xi) SEQUENCE DESCRIPTION: SEQ ID No: 8:
• (2) INFORMATION ON SEQ ID NO: 9:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 18 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
• (2) INFORMATION ABOUT SEQ ID NO: 10:
  • (i) SEQUENCE CHARACTERISTICS:
    • (A) LENGTH: 19 base pairs
    • (B) TYPE: nucleic acid
    • (C) STRAND FORM: Single
    • (D) TOPOLOGY: linear
  • (ii) MOLECULE TYPE: DNA (genomic)
  • (iii) HYPOTHETICAL: NO
  • (iii) ANTISENSE: NO
  • (vi) ORIGINAL ORIGIN:
    • (A) ORGANISM: Staphylococcus aureus
  • (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Claims (10)

1. An oligonucleotide probe that hybridizes with DNA or RNA from methicillin-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. A polynucleotide probe containing DNA or RNA from methicillin resistant staphylococci hybridized, characterized in that they Contains 467 bases or parts thereof and the nucleotide sequence as in Sequence listing SEQ ID No 2 or parts thereof contains or mutates Sequences thereof or labeled sequences thereof. 3. Starter oligonucleotides (primers) for the amplification and the specific Detection of parts of the mec A gene, characterized in that it contains the Nucleotide sequence as contained in the sequence listing SEQ ID No 3 and 4. 4. Hairpin oligonucleotides for RNA amplification and specific Detection of parts of the mec A gene, characterized in that it contains the Nucleotide sequence as contained in the sequence listing SEQ ID No 6 to 8. 5. A method for the detection of methicillin-resistant staphylococci in sample material, characterized in that

• a) oligonucleotide or polynucleotide probes according to claim 1 and 2 for the specific hybridization and detection of methicillin-resistant nucleic acid from staphylococci are used
• b) Oligo and polynucleotide probes are labeled with reporter molecules using known methods
• 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 methicillin-resistant staphylococci via the labeling of the amplified target DNA.

6. A method according to claim 5, characterized in that oligo nucleotide probes according to claim 1 are used. 7. A method according to claim 5, characterized in that a poly nucleotide probe according to claim 2 is used. 8. A method according to claim 5, characterized in that for the Amplification of parts of the mec A gene the primers according to claim 3 be used. 9. A method according to claim 5, characterized in that for the Amplification of parts of the mec A gene hairpin oligonucleotides after Claim 4 are used. 10. Test kit containing one or more of the oligonucleotides according to claims 1 to 4 as well as buffers and other solutions for practical implementation an analysis for methicillin resistance in clinical specimens.