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  • 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
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to a method for the detection of resistant fungal cells in clinical material.
• the general interest in methods for the detection of fungal cells can be seen against the background that fungal species in particular as nosocomial pathogens have gained considerable importance in immunosuppressed patients, in particular in recent years.
• the previously known methods for the analysis of fungal infections primarily aim to enable a diagnosis of the fungal infection and an identification of the pathogenic fungal species. To do this, one uses, for example, a cultivation of fungal species from clinical material on suitable nutrient media and, if necessary, molecular biological methods.
• the genus Candida which belongs to the fungi imperfecti, is one of the medically most important facultative pathogenic fungal genera. It causes the so-called candida mycoses, also called candida.
• candida mycoses also called candida.
• the most important pathogen within the genus Candida is the species Candida albicans, which in addition to the mostly less serious infections of the skin and mucous membranes also causes deep organ mycoses or system mycoses.
• Systemic mycoses are fungal infections that affect not only the skin or mucous membranes, but also other organs, organ systems or even the whole organism. In the latter case one speaks of "generalized" fungal infections.
• Candida albicans infections can be diagnosed is in the publication "Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus pecific gene" by Buchman et al. (1990), Surgery 108, pages 338 to 347.
• a Candida albicans infection is detected by PCR aplification of another fungal-specific gene area, namely the gene for the enzyme 14-oj-lanosterol demethylase.
• PCR products are not detected here by hybridization, but by separation in the agarose gel and staining of the DNA with ethidium bromide.
• molecular biological techniques are used to detect fungal infections in patient material and, as in the Niesters publication to additionally distinguish different species of the genus Candida.
• azole derivatives the polyene amphotericin B, flucytosine, griseofulvin and terbinafine.
• antifungals commonly used are the azole derivatives, which include, for example, the fluconazole.
• azole derivatives are initially given as the "agent of choice", but it can only be concluded that the patient is not infected with azole derivative-resistant fungal cells despite the fact that there is no improvement despite high doses and long-term treatment. This diagnosis is often only made when the course of infection in the patient takes a dramatic course despite treatment with the azole derivatives, which are ineffective here.
• this object is achieved by a method with the following steps:
• fungus-specific nucleic acids can preferably be extracted from blood, but also from biopsy material, sputum, mucosal smears or other patient material. Either the fungus-specific DNA or RNA can be isolated, which can then be detected by DNA / DNA, DNA / RNA or RNA / RNA hybridization.
• hybridization probes used being radioactive or non-radioactive labeled and then the specific hybridization being detected by autoradiography or enzyme-catalyzed color reactions.
• the hybridization probes are directed against a DNA segment from the 14- ⁇ -lanosterol demethylase gene.
• the inventors of the present application were able to show that, in the case of fungal species which have resistance to azole derivatives, mutations occur in this gene region of the fungal DNA, which surprisingly correlate highly significantly with the clinical and microbiological finding of an azole derivative resistance.
• Ergosterol is a steroid that is stored in the so-called plasmalemma, the phospholipid layer that adheres to the inside of the cell wall of the fungi.
• Ergosterol is synthesized in the cell from lanosterol, a precursor.
• the decisive step in the synthesis of ergosterol from lanosterol is catalyzed by the enzyme 14- ⁇ t-lanosterol demethylase, or 14-DM for short. Since ergosterol is an essential component of the plasma membrane, cell division can no longer take place in the absence of ergosterol. A new synthesis of cell wall and plasma membrane is always necessary for cell division.
• the inhibition of ergosterol synthesis which is essential for the growth of the fungal cells, can be successfully used to combat fungal infections.
• hybridization probes are used against the gene which codes for the protein on which the azole derivatives attack directly.
• changes in the nucleic acid sequence occur frequently in this gene.
• the specific hybridization probes are then designed in such a way that they recognize these sequence changes, that is to say only bind to gene sections of fungal cells which have resistance to azole derivatives.
• the hybridization probes are directed against a DNA segment from the 14- ⁇ -lanosterol demethylase gene of the species Candida albicans. This gene is also called ERG16 gene in Candida albicans.
• the advantage here is that the hybridization probes make it possible to diagnose resistant strains of the most widespread pathogenic fungal species, namely Candida albicans.
• a PCR reaction is carried out between steps a) and b), in which sections of the 14-cu-lanosterol demethylase gene are amplified.
• This measure therefore has the advantage that the method gains both sensitivity and specificity, since large quantities of starting material are generated, in which only the required gene segment is specifically contained.
• the material amplified with PCR is then used, for example, in Southern hybridization.
• nucleotide sequences SEQ ID-No: 1 and SEQ ID-No: 2 or the nucleotide sequences SEQ ID-No: 3 and SEQ ID-No: 4 are used as primer pairs in the PCR reaction become.
• these primer pairs are used to amplify DNA sections which contain DNA sequences characteristic of resistant fungus species.
• the resulting amplification products are significantly shorter than the entire gene and thus enable easier processing.
• nucleotide sequences SEQ ID-No: 5 to 8 of the enclosed sequence protocol are used as hybridization probes in step b).
• the inventors of the present application have recognized that surprisingly resistant Candida species, the resistance of which has only come about through a single base exchange in the ERG16 gene, can be distinguished from sensitive strains which do not have this mutation using these hybridization probes.
• the hybridization probes are marked with digoxigenin in step b) and used in the Southern hybridization. Specific hybridization is then detected using enzyme-conjugated anti-digoxigenin antibodies, the enzymes catalyzing color reactions.
• the advantage here is that the labeling of the hybridization probes does not have to be radioactive.
• large amounts of hybridization probes can be labeled at the same time, which can then be aliquoted and stored at -20 ° C because they are stable over a long period of time. Aliquots from the same labeling reaction can then be thawed and used for the individual detection reactions, so that high reproducibility is ensured over long periods of time.
• radioactive labeling methods and other non-radioactive labeling methods, such as labeling with biotin, are also possible.
• nucleic acid to be analyzed can be quickly applied to a membrane, for example a microcellulose or nylon membrane.
• a membrane for example a microcellulose or nylon membrane.
• the fastest method is the "blot-blot” or “dot-blot” method familiar to the person skilled in the art.
• the fungal DNA can be used directly or previously amplified by PCR in the Southern hybridization. However, it is understood that it is also possible to analyze fungal specific RNAs. These can either be isolated directly from the cytoplasm of fungal cells, or can only be produced by reverse transcription. The analysis can then be carried out by Northern blot or further RNA detection reactions.
• the hybridization does not have to be carried out on membranes, such as, for example, in Southern or Northern hybridization, but can also be carried out in solution or on columns.
• step b If the hybridization probes with the nucleic acid sequences SEQ ID-No: 4 to 8 are used in step b), it is preferred if after the hybridization a washing step is carried out at a temperature which is about 1 ° C. below that Melting temperature (Tm) of the hybridization probe used in each case.
• Tm Melting temperature
• the invention further relates to the nucleotide sequences SEQ ID-No: 1 to 8 from the enclosed sequence listing. It is preferred if the nucleotide sequences SEQ ID-No: 1 and 2 are used as primers for the PCR reaction and the nucleotide sequences SEQ ID-No: 5 and / or 6 as hybridization probes in the method for the detection of azole derivative-resistant fungal cells .
• nucleotide sequences SEQ ID-No: 3 and 4 are used as primers and the nucleotide sequences SEQ ID-No: 7 or 8 as hybridization probes in the method according to the invention.
• This measure has the advantage that, in this way, initially easily amplifiable, easy-to-handle DNA fragments of 300-400 base pairs are provided in large quantities in the PCR, and then the base exchanges which may be contained in these PCR fragments with the Hybridization probes can be identified.
• the inventors of the present application were able to show that a number of individual base exchanges occur in azole derivative-resistant fungal strains with respect to the azole derivative-sensitive fungal strains.
• a base exchange from T to G in the ERG16 gene leads to the amino acid phenylalanine No. 105 of the enzyme 14-DM being mutated to a leucine.
• This T / G exchange can be detected at the gene level using the hybridization probe with the nucleotide sequence SEQ ID-No: 5.
• the inventors have recognized that a base exchange from A to C can occur in resistant fungal strains, whereby the amino acid glutamine No. 142 is mutated to a proline.
• This mutation can be detected using the hybridization probe with the nucleotide sequence SEQ ID-No: 6.
• These two point mutations are detected with the hybridization probes with the nucleotide sequences SEQ ID-No: 7 and 8, respectively.
• resistant fungal strains on the specifically amplified PCR fragment either contain one or more of the base changes, it is advantageous if the corresponding hybridization probes are used simultaneously in the Southern hybridization. With a positive signal, there is in any case a resistant fungus species. If only one of the hybridization probes is used for hybridization, it can be demonstrated which mutation has occurred in this resistant fungal strain and whether one or more mutations are present.
• nucleotide sequences SEQ ID-No: 1 and SEQ ID-No: 4 can also be combined as primers, so that then on the amplified PCR fragment of approximately 1,400 base pairs all with the hybridization probes with the nucleotide sequences SEQ ID-No: 5 to 8 detectable mutations are included.
• the invention further relates to a kit for analyzing fungal infections with azole derivative-resistant fungal strains, one or more of the nucleotide sequences SEQ ID-No: 1 to 8 being contained in this kit.
• the kit contains all the solutions required to carry out the PCR reaction and hybridization be included. This makes it possible to have the method according to the invention carried out in a routine laboratory by trained personnel. In addition, the process can be carried out quickly and without lengthy preparations with high reproducibility if all the substances required for many reactions are provided in the kit.
• Candida albicans strains For the analysis and comparison of resistant Candida albicans strains with sensitive Candida albicans strains, patient material or yeast samples are incubated for 48 hours at 30 ° C on a medium used for yeast cultivation, the Sabouraud glucose agar. Then several colonies are inoculated and taken up in sterile 0.9% sodium chloride solution.
• the fungal cells are disrupted by alkaline lysis (50 mM NaOH, 10 minutes, 95 ° C.) and then neutralization and enzymatic treatment with zymolyase from Sigma.
• the proteins are denatured in Tris / EDTA and 10% SDS solution at 65 ° C.
• the now available solution contains debris from the fungal cells and free fungal DNA, which must now be isolated.
• a protein precipitation with 5 M potassium acetate is carried out and the DNA is precipitated by adding ice-cold isopropanol.
• the precipitation product is used for the further process steps.
• Example 3 Amplification of a DNA fragment from the
• the PCR reaction first serves to amplify sections from the ERG16 gene to which the specific hybridization probes bind.
• the ERG16 gene which is 1851 bp long, is divided into sections that are easy to handle and that can be easily amplified in PCR.
• DNA sequence SEQ ID-No: 5 and / or 6 is to be used as the hybridization probe, a PCR with primers with the nucleotide sequences SEQ ID-No: 1 (upstream primer) and 2 (downstream primer) is carried out.
• a PCR product is then obtained with the primers mentioned, which comprises the range from base 379 to base 676, that is to say approximately 300 base pairs of the ERG16 gene.
• the PCR primers with the nucleotide sequences SEQ ID-No: 3 (upstream primer) and 4 (downstream primer) are used.
• the PCR conditions are as follows:
• Terminal extension 5 min at 72 ° C
• the high magnesium concentration in the buffer ensures a high specificity of the polymerase, which can work at 72 ° C in the extension step at its optimum temperature.
• the PCR reactions result in a sufficient quantity of starting material to be used to further analyze whether the DNA comes from resistant or sensitive fungal cells.
• the PCR products obtained in Example 3 are heat-denatured and applied to nylon membranes, for example in the slo-blot process familiar to the person skilled in the art.
• the DNA is cross-linked on the nylon membrane.
• the hybridization probes are labeled by incorporating digoxigenin-labeled nucleotides into methods which are known to the person skilled in the art (for example “nick translation” or random priming ”).
• the membrane is prehybridized for 20 minutes in 6 x SSPE, 5 x Denharts solution, 0.1% N-lauryl-sarcosine-Na, 0.02% SDS at 42 ° C.
• hybridization is then carried out in the prehybridization solution described above, to which 30 pM digoxigenized hybridization probe has been added, for 20 minutes at 42 ° C.
• 30 pM digoxigenized hybridization probe has been added, for 20 minutes at 42 ° C.
• the specificity of the hybridization is determined by washing steps which then take place.
• the first two washing steps are carried out for 5 minutes in 2 x SSPE, 0.1% SDS at 42 ° C.
• two washing steps are carried out for 7 minutes each in 6 x SSPE, 1% SDS, the washing temperature being approximately, preferably exactly 1 ° C. below the Tm value.
• the melting temperatures of the hybridization probes and the base changes detectable by the hybridization probes are given in Table I. If the nucleotide sequence of the hybridization probe does not exactly match the corresponding sequence of the PCR fragment, the hybridization probe is washed away in this step.
• the detection reaction is then carried out, in which it is examined whether digoxigenized hybridization probe is present on the membrane or not. This is done in a process according to the manufacturer's protocol of Boehringer Mannheim with the aid of enzyme-conjugated anti-digoxigenin antibodies. The enzyme then catalyzes a reaction that leads to the production of an insoluble color complex.
• the patient contains fungal cells which are resistant to azole derivatives.
• therapy with azole derivative antifungals is therefore pointless and the therapy for combating the Candida infection must be changed.

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• 1996
  • 1996-10-22 DE DE19654946A patent/DE19654946A1/de not_active Withdrawn
• 1997
  • 1997-10-04 AU AU47074/97A patent/AU731424B2/en not_active Ceased
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