WO2002027021A2

WO2002027021A2 - Method for detecting fungal infections

Info

Publication number: WO2002027021A2
Application number: PCT/EP2001/011023
Authority: WO
Inventors: Erik C. BÖTTGER; Jens Rosenau; Phillip Kirschner; Thomas Jack
Original assignee: Cytonet Gmbh & Co. Kg
Priority date: 2000-09-26
Filing date: 2001-09-24
Publication date: 2002-04-04
Also published as: DE10048009A1; WO2002027021A3; WO2002027021A8

Abstract

The invention relates to a method for detecting clinically relevant fungal infections. A multiplex amplification reaction is carried out using DNA that has been isolated from patient material in order to detect clinically relevant fungal infections of the <i>Candida</i> and <i>Aspergillus</i> species, a region of the 18s RNA gene of the pathogen being amplified as the target. Only sequences of clinically relevant species are amplified.

Description

Procedure for the detection of fungal infections

The present invention is in the field of detection of pafhogenic fungal infections using PCR.

State of the art

Fungal infections in healthy people or non-immunosuppressed patients are usually local superficial skin, skin, and nail infections with no serious health effects. However, fungal infections that occur in immunosuppressed people, for example after transplants or in HIV patients, are always life-threatening. For this reason, in these immunosuppressed patients, if there is a long-standing fever of unknown cause, a fungal infection is also assumed without pathogen detection and treated for safety reasons (Lortholary and Dupont, B., Clin. Microbiol. Rev. 10: 477-504, 1997). To avoid unnecessary or incorrect therapeutic measures, a simple, safe and sensitive diagnosis of a systemic fungal infection is therefore desirable.

The spectrum of possible fungal pathogens from a disseminated fungal infection is wide. However, infections with predominantly endemic obligatory pathogens of primary system mycoses such as Histoplasma capsulatum, Coccidoides immitis, Blastomyces dermatitidis or Paracoccidoides brasiliensis are rather the exception. On the other hand, infections caused by the genera Candida and Aspergillus are of increasing importance in the case of invasive or disseminated fungal infections, so that a reliable rapid detection is desirable, especially for these genera.

In this context, the Candida species C. albicans, C. guilliermondii, C. maltosa, C. parapsilosis, C. tropicalis, C. viswanatii, C. glabrata and C. krusei are of particular clinical relevance in this context. The species A. fumigatus, A.niger, A. nidulans, A. flavius and A. terreus are of particular importance as Aspergillus germs.

Both Candida and Aspergillus fungal infections can be detected, for example, by detecting disseminated fungal cells in the blood as units forming colony on culture dishes (CFU = colony-forming units = PFU). The number of fungal cells to be detected is usually in the range of 1-10 colony-forming units per ml of blood (Kiehn, Eur. J. Clin. Microbiol. 8, 832-837, (1989)). For a mushroom proof is therefore an enrichment of the fungal cells is an advantage. However, an analysis of PFUs in cells enriched from whole blood is complex and time-consuming.

Alternatively, methods based on the amplification of nucleic acid sequences of infectious germs with the aid of PCR (U.S. 4,683,195, U.S. 4,683,10) represent a sensitive possibility for the detection of infections. The identification of the correct amplification products takes place either with the aid of gel electrophoresis or with the aid of suitable hybridization probes. Processes have recently become established,. in which an amplification reaction can be detected in real-time mode and followed on-line (WO 97/46707, WO 97/46712, WO 97/46714).

For the identification of species based on nucleic acid sequences, methods have proven to be particularly suitable in which ribosomal RNA sequences are analyzed (EP 0 131 052). This is mainly due to the fact that the ribosomal RNA genes occur in the genome in high copy numbers and can be detected with a comparatively high sensitivity. In addition, RNA genes are particularly suitable for systematic or taxonomic analyzes because the corresponding genes, for example the 18 s RNA genes, both have regions that are highly conserved in evolution and regions that are hypervariable within a genus, for example.

Several methods based on the analysis of 18s RNA sequences have also been published for Candida and Aspegillus (Hopfer et al. Med. Vet. Mycol. 31: 65-75 (1993)), Maldmura et al., J. Med. Microbiol. 40: 358-364 (1994) Einsele et al. J. Clin. Microbiol. 35: 1353-1360 (1997)., With the help of which the identity of a pathogenic pathogen can be established. However, all of the methods are only suitable for the detection of a single species, so that the entire spectrum of Indian-relevant fungal infections can only be covered with the aid of a large number of different reactions.

WO 97/07238 describes a method for the detection of fungal cells in clinical material, in which initially integrated fungal cells from whole blood are enriched. The disadvantage of the method described in WO 97/07238, however, lies primarily in the fact that the primers used in a background of human genomic DNA obviously lead to unspecific amplification products and can only be used specifically for the detection of indinically relevant fungal species if the amplification reaction is complex Sample preparation, ie isolation of intact fungal cells, preferably from whole blood. In summary, the current methods known from the prior art for the timely diagnosis of such an infection are limited in terms of their experimental complexity, their speed, specificity and sensitivity. In particular, at the time of the invention there was no method with which a fungal infection with high specificity could be detected.

The object of the present invention was therefore to provide a fast, simple and sensitive method with which. all Hinisch relevant fungal infections of the genera Candida and Aspergillus can be detected.

Brief description of the invention

The object is achieved by a multiplex amplification reaction for the detection of only clinically relevant fungal infections of the genera Candida and Aspergillus, in which a region of the 18s RNA gene of the pathogen is amplified as the target, characterized in that only sequences of pathogenic Candida and Aspergillus species are amplified.

This object is achieved, in particular, by a multiplex amplification reaction for the detection of Indian relevant fungal infections of the genera Candida and Aspergillus, in which a region of the 18s RNA gene of the pathogen is amplified as the target, so that sequences of the species C. albicans, C. guiUiermondii, C. maltosa, C. parapsilosis, C. tropicalis, C. viswanathii, C. glabrata, C. crusei, A. fumigatus, A.niger, A. nidulans, A. flavus, A. terreus, A. awamori, A. oryzae, A.parasiticus, A. soenthalte, A. tamarii, A. sojae, A. wentii, A.restrictus, A. ochraceus, A. clavatus and A. candidus are amplified.

In the event that aspergillosis is unlikely due to other clinical indications, the method according to the invention consists of a multiplex amplification reaction for the detection of clinically relevant fungal infections of the genus Candida, in which a region of the 18s RNA gene of the pathogen is amplified as the target, sequences of the Species C. albicans, C. guiUiermondii, C. maltosa, C. parapsilosis, C. tropicalis, C. viswanatii, C. glabrata and C. krusei can be amplified.

Advantageously, exactly one upstream primer is used in these methods, the sequence of which corresponds to a specific sequence of the 18 sRNA gene, which in all species to be detected is identical. Such a primer is usually 13-25 nucleotides in length. The sequence of the primer is preferably a continuous partial sequence of a sequence according to Seq. ID. No. 1.

Primers with a length of 13-25 nucleotides have proven to be suitable as downstream primers, the sequence of which is a continuous partial sequence of a sequence according to Seq. ID. No. 2, Seq. ID. No. 3 or Seq. ID. No. 4 or Seq. ID. No. 5. The invention thus relates in particular to methods in which one, two, three or all downstream primers with partial sequences in accordance with. Seq. Id. No 2-5 can be used.

It has proven to be advantageous if the product of the ampHfication reaction is additionally detected by hybridization. In a particular embodiment of the method according to the invention, hybridization probes are used whose sequences are identical to or complementary to a partial sequence of all 18s RNA sequences which were amplified during the amphication reaction. The invention thus also relates to hybridization probes with a length of 15 to 100 and particularly preferably with a length of 18-35 nucleotides, which have a continuous sequence according to Seq. ID. No. 6 included as well as methods in which such probes are used.

In a special embodiment of the invention, the amplification products are detected with the aid of fluorescence detection, with the hybridization probes generally being labeled with a fluorophore. Embodiments in which the amplification product with the aid of fluorescence resonance energy transfer (FRET ) is proven. Two fluorophores-labeled hybridization probes are used, which hybridize to adjacent but not overlapping sequences of the target nucleic acid in such a way that when the probes are hybridized to the target molecule, the two fluorophores are brought into spatial proximity to one another. A probe is labeled with a fluorescence resonance energy donor such as fluorescein. The other probe is labeled with a fluorescence resonance energy acceptor such as LC-RED-640 or LC-RED-705 (Röche Molecular Biochemicals). Preferred for the detection of Candida and Aspergillus are two hybridization probes to be used as a FRET pair according to Seq. ID. No. 7 and 8.

Finally, the present invention also relates to kits for carrying out the methods according to the invention. Brief description of the pictures

Figure * go 1:

Schematic representation of a Candida 18s RNA gene with primers for an inventive multiplex reaction for the detection of Candida. VI -V8 denote variable regions of the 18s RNA gene. F3 denotes the position of the forward primer with SeQ: Id. 1. F16, F19 and F21 denote the positions of the three reverse primers with SEQ. Id. No.2-4. “Hybridization probe” denotes a probe according to Seq. Id. No. 6.

Figure 2:

Schematic representation of an Aspergillus 18s RNA gene with primers for the detection of Aspergillus. According to the invention, the primer F15 (Seq. Id. No. 5) is used together with the Candida primers according to Figure 1 in a multiplex reaction. Again: V1-V8 denote variable regions of the 18s RNA gene. F3 denotes the position of the forward Primer with Seq: Id. No. 1. F15 denotes the position of the reverse primer with SEQ. Id. No. 5. "Hybridization probe" denotes a probe according to Seq. Id. No. 6.

Detailed description of the invention

The invention initially relates to a multiplex amplification reaction for the detection of only clinically relevant fungal infections of the genera Candida and Aspergillus, in which a region of the 18s RNA gene of the pathogen is amplified as the target.

Sequences of the species C. albicans, C. guiUiermondii, C. maltosa, C. parapsilosis, C. tropicalis, C. viswanatii, C. glabrata, C. krusei, A. fumigatus, A.niger, A. nidulans, A flavus, A. terreus, A. awamori, A. oryzae, A.parasiticus, A. soentten,. tamarii, A. sojae, A. wentii, A.restrictus, A. ochraceus, A. clavatus, A. candidus and preferably only amplified sequences of the species mentioned. Thus, all clinically relevant species of the two genera mentioned are included, which occur with a certain frequency in everyday clinical practice and must be treated due to their pathogenic effects. In contrast, species that frequently occur as contamination in laboratories are not detected by the method according to the invention. The detection of non-pathogenic

Species of this and other genera by the method according to the invention, however, cannot be completely excluded.

Under certain clinical conditions, aspergiUosis can be excluded, and it is only necessary to test the immunosuppressed patient for a pathogenic Candida infection. In this FaU, the method according to the invention consists of a multiplex amplification reaction for the detection of clinically relevant fungal infections of the genus Candida, in which a region of the 18s RNA gene of the pathogen is amplified as the target, sequences of the species C. albicans, C. guilliermondii, C. maltosa, C. parapsilosis, C. tropicalis, C. viswanatii, C. glabrata and C. krusei and preferably only sequences of the species mentioned are amplified.

Prerequisite for carrying out the method according to the invention is firstly DNA isolation from patient material, the patient material preferably being the patient's blood. In principle, all methods known from the prior art can be used for this purpose. Optionally, the mushroom cells can be enriched in a first step (WO 97/07238). Alternatively, other body material such as tissue, stool, urine or sputum can be examined in some rots.

To isolate the fungal DNA, a lysis of the fungal cells is generally required, which can also be carried out by various methods known to the person skilled in the art or a combination of methods known to the person skilled in the art. For example, a method has proven to be particularly effective for this, in which the mushroom cells are mechanically comminuted after digestion by the enzyme lyticase and lysis by SDS at 65 ° C. using glass particles.

The amplification reaction of the respective 18s rRNA target sequence is preferably carried out using a conventional PCR reaction under standard conditions using Taq polymerase or other thermostable DNA polymerases. ι Chemically synthesized deoxy-ribonuldeotides are generally used as amplification primers. In principle, however, other nucleic acid molecules or theirs can also be used

Derivatives such as PNA (Peptide Nucleic Acids) can be used. In addition, primers according to the invention can also be used with detectable or immobilizable

Molecules such as biotin or digoxygenin can be conjugated. As primers for a PCR reaction, at least one sogenanter upstream primers and a downstream primer sogenanter is required, wherein the sequence of the upstream Primes in 5 corresponds to the coding sequence of the target nucleic acid ^£ ~ 3 'orientation. The sequence of the downstream primer corresponds to an antisense sequence in 5 '-3' orientation. According to the invention, however, not only two primers are used, but rather several primers in the sense of a so-called multiplex reaction. This makes it possible to amplify not only sequences of a single species, but also sequences of several species in one reaction mixture. ,

Advantageously, exactly one upstream primer is used in these methods, the sequence of which corresponds to a specific sequence of the 18 sRNA gene, which is identical in all species to be detected. Such a primer is usually 13-25 nucleotides in length. The sequence of the primer is preferably a continuous partial sequence of a sequence according to Seq. ID. No. 1.

In the context of the present invention, a continuous partial sequence is understood to mean the partial sequence of a sequence which is identical to the respective sequence without any insertions or internal deletions, but can be shortened at one or both ends. A continuous part sequence in the sense of the invention can, however, also correspond to the entire respective sequence.

Primers with a length of 13-25 nucleotides have proven to be suitable as downstream primers, the sequence of which is a continuous partial sequence of a sequence according to Seq. ID. No. 2, Seq. ID. No. 3 or Seq. ID. No. 4 or Seq. ID. No. 5 figure. Primer with partial sequences according to Seq. Id. No. 2-4 are used for the amplification of Candida sequences. A primer according to Seq.Id. No. 3 is essential for the detection of C. glabrata; a primer according to Seq. Id. No. 4 is required for the detection of C. Krusei. Primer with partial sequences according to Seq. Id. No. 5 are used to amplify AspergiUus sequences. The invention thus relates in particular to methods in which one, two, three or three external primers with partial sequences in accordance with. Seq. Id. No 2-5 can be used.

The AmpUfikationsprodukte can be detected by different methods. A classic detection method is agarose gel electrophoresis, in which the PCR fragments are separated according to size and thus identified. Another alternative is the so-called PCR-ELISA (Röche Molecular Biochemicals Catalog). In this method, at least one primer labeled with an immobilizable group such as, for example, biotin is used, so that, following the PCR reaction, the amplification product can be bound to a solid phase, such as an ELISA plate, which is coated, for example, with streptavidin as a binding partner for biotin ,

In external cases, however, it has proven to be advantageous if the product of the amplification reaction is additionally detected by hybridization. This results in a higher sensitivity of the assay, so that purge infections can be detected even if the titer of disseminated tumor cells in the blood or in other patient material is only very low.

The hybridization probes suitable for this preferably have a length of 15 to 100 and particularly preferably a length of 18-35 nucleotides. They are thus also the subject of the invention provided that they have a continuous sequence according to Seq. ID. No. 6 included. For the purposes of this invention, this is understood to mean all sequences which have a sequence section with the entire sequence of Seq. Id. No. 6 included without any deletion or insertion. In addition, however, ^this' probes may also contain additional sequence segments. Such probes preferably hybridize with external amplification products which can be amplified using the primers according to the invention. The invention also relates to methods in which the probes described are used

As a rule, chemically synthesized deoxy-ribonucleotides are also used as probes, which can optionally be replaced by other nucleic acid molecules or their derivatives such as, for example, PNA (Peptide Nucleic Acids). By definition, these probes are conjugated to a detectable label. This can be, for example, a fluorescent dye, an enzyme, a radioactive atom or a group that can be detected by mass spectrometry. Depending on the exact detection format, the label can be introduced on any ribose or phosphate group of the oligonucleotide. Markings at the 5 'and 3' end of the nucleic acid molecule are preferred.

In a special embodiment of the invention, the amplification products are therefore detected by means of fluorescence detection, the hybridization probes being labeled with a fluorophore. The amplification products can be particularly preferably used in real time PCR monitoring

(WO 97/46707) can be detected. Various homogeneous test procedures are possible in which either at least one fluorescence-labeled hybridization probe or a fluorescent DNA binding dye is already in the sample to be analyzed during the amplification:

(i) DNA binding dye

In this case, the respective amplification product is detected by a DNA binding dye which, when interacting with double-stranded nucleic acid, emits a corresponding fluorescence signal after excitation with light of a suitable wavelength. The dyes SybrGreen and SybrGold (Molecular Probes) have proven to be particularly suitable for this application. Alternatively, intercalating dyes can also be used.

(ii) TaqMan hybridization probes

A single-stranded hybridization probe is labeled with 2 components. When the first component is excited with light of a suitable length, the absorbed energy is transferred to the second component, the so-called quencher, according to the principle of fluorescence resonance energy transfer. During the annealing step of the PCR reaction, the hybridization probe binds to the target DNA and is degraded by the 5'-3 ^c exonuclease activity of the Taq polymerase during the subsequent elongation phase. As a result, the excited fluorescence component and the quencher are spatially separated from one another, so that a fluorescence emission of the first component can be measured.

(iii) Molecular Beacons

These hybridization probes are also labeled with a first component and a quencher, the markings preferably being located at the two ends of the probe. In solution, both components are in close proximity to each other due to the secondary structure of the probe. After hybridization to the target nucleic acid, the two components are separated from one another, so that after excitation with light of a suitable wavelength, the fluorescence emission of the first component can be measured (Lizardi et al., US 5,118,801). (iv) FRET hybridization probes

For this test format, 2 single-stranded hybridization probes are used at the same time, which are complementary to neighboring stands of the same strand of the amplified target nucleic acid. Both probes are labeled with different fluorescent components. When a first component is excited, it transfers the absorbed energy to the second component according to the principle of fluorescence resonance energy transfer, so that when both hybridization samples bind to the target molecule, a fluorescence emission of the second component can be measured (WO 97/46707).

As a so-called donor component, for example, fluorescein is excited with light of a suitable length. If there is spatial proximity to a suitable acceptor component such as, for example, certain rhodamine derivatives, resonance energy transfer then takes place to the acceptor component, so that the acceptor molecule emits light of a specific emission wavelength.

The hybridization probes can be marked using standard methods at the 5 'end, at the 3 ^C end or internally. The two oligonucleotide probes can preferably hybridize to the same strand of the target nucleic acid, one dye preferably located on the 3 ^c -terminal nucleotide of the first probe and the other dye preferably located on the 5 'terminal nucleotide of the second probe, so that the Distance between the two is only a small number of nucleotides, which number can be between 0 and 30. When using fluorescein in combination with a rhodamine derivative such as, for example, LC-RED-640 or LC-RED-705 (Röche Molecular Biochemicals), distances of 0-15, in particular 1-5 nucleotides and in many cases of a nucleotide have been found advantageous out.

While maintaining the nucleotide spacings between the dye components, it is also possible to use probes that are not terminally but internally conjugated to one of the dyes. In the case of double-stranded target nucleic acids, probes can also be used which bind to different strands of the target, provided that a certain nucleotide spacing of 0 to 30 nucleotides between the two dye components is maintained. (Bernard et al, Analytical Biochemistry 235, p. 1001-107 (1998)).

The use of such FRET pairs for the detection of amplification products during or after a multiplex amplification reaction has proven particularly advantageous. This enables parallel "real-time monitoring" of PCR reactions, wherein data for generating the amplification product can be determined as a function of the number of reaction cycles that have been carried out. This is usually done by hybridizing the oligonucleotides of the FRET pair to the target nucleic acid as a result of the reaction and temperature conditions during the necessary annealing of the amplification primer to the nucleic acid to be detected, and emitting a correspondingly measurable fluorescence signal with suitable excitation. On the basis of the data obtained, the amount of the target nucleic acid originally used can thus be determined quantitatively.

In another preferred embodiment, the multiplex amplification products are detected after the amplification reaction has ended, the temperature being continuously increased in the course of a melting curve analysis after hybridization of the FRET pair to the target nucleic acid to be detected. At the same time, the fluorescence emitted as a function of the temperature is measured, and in this way a melting temperature is determined at which the FRET pair used no longer hybridizes to the sequence to be detected. If mismatches occur between the FRET pair used and the amplification product, the melting point is significantly lowered. In this way, different target nucleic acids can be identified with a FRET pair, the sequences of which differ slightly from one another by one or a few point mutations, so that the identity of a specific generated amplification product can also be confirmed with a melting point analysis.

For the detection of Candida and Aspergülus, two hybridization probes according to Seq. ID. No. 7 and 8 proved to be suitable. The first probe with a sequence acc. Seq. Id. No. 7 is labeled according to the invention with a fluorescence resonance energy donor such as fluorescein. The second probe is labeled with a fluorescence resonance energy acceptor such as LC-RED-640 or LC-RED-705 (Röche Molecular Biochemicals) and has a sequence according to Seq. Id. No. 8th.

Finally, the present invention also relates to kits for carrying out the methods according to the invention. These kits preferably contain primers with sequences according to Seq. ID. No. 1-4 or Seq. ID No. 1-5 and optionally other generic reagents for the amplification of nucleic acids or the detection of PCR amplification products. These kits particularly preferably also contain hybridization probes according to the invention, for example with sequnces according to Seq. Id. No.6,7 and 8. As generic reagents can these kits contain, for example, thermostable DNA polymerases, deoxynucleotides and suitable buffer substances.

As already stated, a DNA isolation procedure with several steps is necessary for the detection of puddles at the genomic level. It has been shown that, despite strict application of measures to avoid contamination during sample processing with the methods in question, false positive signals always appear in the laboratory due to the ubiquitous occurrence of pore spores or DNA in the environment and in the used ones Reagents, a universeUes Püz detection method is not suitable for routine diagnostics.

Because of these problems, the invention consists of a PCR detection system with a reduced spectrum, which is limited to the detection of clinically important fungi and is free from contamination problems. In the search for suitable oligonucleotides in the area of the 18S rRNA, which as far as possible detect human pathogenic fungi, problems usually arise as soon as oligonucleotides are used for the amplification which contain several Püzgenera in the spectrum. Since the human pathogenic species of the Candida genus are only distantly related phylogenetically (there is no signature sequence for the Candida genus, which does not include other genera), contaminations always occur when amplified with oligonucleotides that also contain a Candida species in the spectrum Negative controls on. Only the reduction of the spectrum of the PCR to the phylogenetically related species with C. albicans results in contamination-free negative controls, for example when using primers according to Seq. Id. No. 1 and 2. However, the spectrum lacks the two important pathogens C. krusei and C. glabrata, which are phylogenetically only relatively distantly related to Candida albicans.

Since there are no other sequence regions within the 18S rRNA for the derivation of suitable oligonucleotides, the spectrum of which includes these two species, C krusei and C. glabrata must each use a species-specific PCR be amplified. According to the invention, this is done using PCR reactions

Primers according to Seq. Id. Nos. 1 and 3 or 1 and 4.

The system with the broadest spectrum of human pathogenic fungi for the detection of Candida or Aspergiülus infections, which no longer shows false positive results due to contamination of the reagents, consists of four individual amplification reactions, which act as a forward primer on the same universe primer in accordance with. Seq. Use 1. In addition to the three reverse primers with Seq. Id. No. 2-4 is a primer according to Seq. For an additional amplification reaction. Id. No. 5 used for the detection of pathogenic Aspergillus species. These four amplification reactions are due to the use of a single forward primer. Seq. Id. No. 1 can easily be combined in a multiplex PCR in a reaction vessel.

Since the sensitivity of a multiplex PCR in the ELISA-PCR method often decreases with the number of oligonucleotides used, in a particular embodiment the ampHfication was done in two PCRs taking place in separate reaction vessels:

• A multiplex PCR comprises the genus Candida: with primers according to Seq. Id. No. 1-4 The reverse primer according to Seq. Id. No. 2, which is specific for the largest part of the Candida genus (C. albicans, C. maltosa, C. parapsilosis, C. tropicalis, C. viswanaihii and others) is treated with the species-specific primers according to Seq. Id. No. 3 (C. glabrata), and 4 (C. krusei) combined (Fig 1). With this multiplex PCR the most important human pathogenic Candida species can be amplified. False positive negative controls of the DNA preparation method do not occur when ampUfilcation with these oligonucleotides.

• The detection of Aspergillus puddles takes place in a separate reaction with PCR primers according to Seq. Id. No. 1 and 5. With this PCR all previously sequenced (18S rDNA) human pathogenic Aspergillus species can be amplified (A. fumigatus, A. niger, A. nidulans, A. flavus, and A. terreus and others). Positive samples are preferably identified by hybridization, for example in ELISA format with a universe probe according to Seq. Id. No. 6 or in real time PCR mode with a pair of hybridization probes according to Seq. Id. No. 7 and 8. Depending on the experimental task control, Aspergillus or Candida, genus-specific or species-specific oligonucleotides can also be used for both methods according to the invention.

The sensitivity of the overall procedure can be determined with dilution series of C. albicans, the number of which was determined microscopically. For this purpose, the cells are added to 10 ml of blood and the entire process, consisting of a corresponding sample preparation and a PCR according to the invention, is carried out.

After hybridization in the PCR-ELISA format, there is usually a detection limit of approx. 3 cells per 10 ml blood. The detection limit in the Lightcycler PCR format with FRET hybridization probes is usually 20 ZeUen per 10 ml blood.

Examples:

Example 1: Processing of blood samples <

5-10 ml of peripheral venous blood were drawn into tubes containing the anticoagulant EDTA using a standard blood collection system. A microscopic number of C. albicans cells were added to blood samples that were used as positive controls to investigate the sensitivity of the detection method. After being transported to the test laboratory, the blood was transferred to a centrifugation vessel with a volume of 50 ml and, after adding 30 ml of a 2% SDS solution, shaken for 5 seconds and left at room temperature for 10 minutes. In this step, all cellular blood components are lysed with the exception of the fungal cells. In a subsequent centrifugation step (30 min., 6000 xg, 15 ° C), the purge cells were separated from the other blood components. After the supernatant had been poured off, the PeUet was taken up by resuspending in 1 ml of water and in a 1.5 ml Reaction tube transferred with screw cap. After centrifugation for 10 minutes at 9,000

RPM (7150 x g) the supernatant was removed, 1 ml of water was added, centrifuged again as before and the supernatant was removed. In the peuet of the reaction vessels are oUe

PüzzeUen concentrated in pure form from the blood used. Human nuldeinic acid and other blood components that can inhibit the sweeping enzymatic reactions have been completely removed by the treatment described above.

Example 2. Fungal nucleic acid isolation

90 μl of water, 10 μl of lOx buffer (0.5 M Tris-HCl pH 7.5; 0.1 M EDTA; 10% Triton X-100), 1 μl of lyticase solution (25,000 units / ml) added and incubated at 37 ° C for 45 min. After the addition of 15 μl of 10% SDS solution containing lyticase (Sigma, final concentration (25,000 U / ml), the samples were incubated for 45 min at 65 ° C. and after the addition of 30 μl glass beads (100 μm diameter), 2 μg salmon sperm DNA,

330 μl of water, 400 μl of phenol / chloroform / iso-amyl alcohol (volume 24/24/1) shaken for 3 minutes in a high-frequency vibrator (Mickle Laboratory, GomshaU, UK). After centrifugation for 10 minutes, the upper phase was removed and, after addition of 24 μl of 3 M sodium acetate and 500 μl of iso-propanol, precipitated in a new vessel within 2 hours at -20 ° C. After centrifugation for 15 minutes at 15,000 rpm, the supernatant was removed and the PeUet with 800 μl 70%

Washed ethanol twice (add ethanol, swirl lx, centrifuge at 15,000 rpm for 10 min and remove supernatant). Then the one isolated in the PeUet

Removed the remaining traces of ethanol in a Speedvac centrifuge and in

75 μl of water added. 15μl of each of these nucleic acid preparations were added to the

Give Aspergülus-specific PCR and the Candida-specific PCR.

Example 3: Multiplex PCR and detection in ELISA format

The following approach was prepared for a multiplex PCR according to the invention in the PCR ELISA format: Approach for Candida multiplex PCR primer acc. Seq. ID. No. 1 biotinuized: 2.0 μl (12.5 pmol) primer acc. Seq. ID. No. 2 0.5 μl (12.5 pmol)

Primer acc. Seq. ID. No. 3 0.5 μl (12.5 pmol)

Primer acc. Seq. ID. No. 4 0.5 μl (12.5 pmol)

Primer acc. Seq. ID. No. 5 0.5 μl (12.5 pmol) dNTP mix 12 μl lOx buffer 7.5 μl

Taq 0.35 ul

DNA 15 ul

H ₂ O ad 75 ul

The DNA was amplified over 40 cycles in an Ericomp thermal cycler denaturation: 10 seconds at 96 ° C;

Annealing: 120 seconds at 54 ° C;

Extension: 120 seconds at 72 ° C.

A probe according to Seq. Id. No. 6 used. The hybridization with the abovementioned OHgonucleotides was carried out using magnetic particles which are labeled with digoxigenin: These Dynabeads (Db) were used in accordance with the information provided by the manufacturer (Dynal). The amplified DNA was bound to the beads according to the manufacturer's instructions: After washing 10 μl of Db suspension twice with “binding and washing buffer” (BW buffer), the Db were taken up in 50 μl of BW buffer and in 10 microtiter plates μl PCR product combined and incubated for 20 min. After washing once with BW buffer, 100 μl NaOH (0.1 M) were added, incubated for 10 minutes and washed with 100 μl TE buffer. The hybridization was carried out after the addition of 6 pM of the corresponding digoxigenized oligonucleotide in 50 μl hybridization solution over 50 min at 47 ° C. Unbound hybridization probe was washed away in three steps with 4x SSC, 0.1% Tween 20 at 52 ° C. Then 50 μl AK reagent was added, incubated at RT for 30 min, washed twice with PBS, 0.05% Tween and 50 μl substrate buffer added. After 90 minutes, the substrate conversion was measured using the fluorescence in a UV ELISA reader at 355nm / 460nm (excitation / emission). Samples that exceeded the mean of the negative controls carried by more than five times the standard deviation of the negative controls were rated as positive. The following solutions were used for this ProtokoU:

d-NTP mix for PCR 2x950 μl H ₂ O

2x12.5 ul dATP 100mM

2x12.5 ul dTTP 100mM

2x12.5 ul dCTP 100mM

2x12.5 ul dGTP 100mM

IQx PBS buffer 2g KCl ^' 2g KH ₂ PO ₄

80 g NaCl 21.6 g Na ₂ HPO. 7 H ₂ O add 101. H ₂ 0.

TE buffer:

Tris 10 mM, pH 7.5 and 8.0, respectively

EDTA 1 mM

B W - buffer (binding + washing buffer) according to Dynal ProtokoU

5mM Tris HCL pH 7.5 0.5mM EDTA

1.0 M NaCL

20 x SSC

3 M NaCl

0.3 M Na citrate (2-hydrate)

Hybridization solution:

5x SSC,

5x Denhardt's,

0.1% Tween 20,

100 µg / ml tRNA

6 pmol digoxigenized oligonucleotide / 50 μl solution Ak labeling (AK antiDIG reagent)

Ak 1: 5000 (Boehringer 1093274) diluted in 1% BSA in PBS

Substrate buffer:

4-Methyl-UmbeUiferylphosphate 0 mg / liter (Sigma 3368-04-5 M 8883)

100 mM Tris-HCl, pH 9.6, 50 mM MgCl ₂

100 mM NaCl.

A large number of püz species were examined in this way. However, amplification products could only be detected in those DNA preparations which contained DNA from pathogenic fungal species of the genera Candida or Aspergülus.

Example 4: Real Time Multiplex PCR

The nucleic acids were isolated as described in Examples 1 and 2. However, only 5 -10 μl DNA was used in the PCR. The multiplex-PCR for the detection of pathogenic Candida puddles was carried out in the LightCycler system (Röche Molecular Biochemicals) according to the information of the manufacturer. The amphfication product was detected after two different protocols, either with the help of SybrGreen as a double-stranded DNA-binding agent or alternatively with the help of FRET hybridization probes. All primers and hybridization probes used were HPLC-cleaned and were stored in Stocl solutions of 100 pM / μl (primer) or 3 pM / μl (probes).

Evidence with SvbrGreen:

2.0 μl FastStart Buffer DNA-Master SYBR Green (Röche MolecularBiochemicals, containing

Buffer, heat-activated Taq DNA polymerase, dNTPs, MgCI ₂ and SYBR Green I

Dye) 10 pM per primer used in accordance with Seq Id No 1, 2, 3 and 4 3.2 μl 25mM MgCl ₂ (working concentration 5 mM) 5 μl sample DNA 20 μl total mixture Detection with hybridization probes:

The hybridization probes used for the detection were labeled according to standard protocols. An oligonucleotide according to Seq.Id. No. 7 labeled with fluorescein at the 3 'end and an oligonucleotide according to Seq. Id. No. 8 marked with LcRed 640 at the 5 'end.

2.0 μl FastStart DNA-Master for Hybridization Probes (Röche Molecular Biochemicals, containing buffer, heat-activated Taq polymerase, dNTPs. And MgCl ₂ ) 5 pM per primer used according to Seq Id. 1-5 2 pM per hybridization probe used according to Seq Id No 7 and 8 3.2 μl 25 mM MgCl ₂ (final concentration 5 mM) 5 μl sample DNA 20 μl total mixture

Both types of approaches were run with the following PCR program and concluded with a melting curve.

The temperature cycles of the PCR program were:

95 ° C 600 sec before the first cycle

95 ° C 10 sec

50 ° C 10 sec

72 ° C 3O sec each for 55 cycles

The melting curves were after a brief denaturation at 95 ° C in an IntervaU from 65 ° C to 95 ° C in 0.2 ° C steps with continuous fluorescence measurement on channel 1 (SYBR Green), channel 2 (LC-Red 640) or Channel 3 (LC-Red 705) with the help of LightCycler Software 3.0 is available.

In the FRET hybridization probe format, the sensitivity was checked with 10 ml blood samples to which C. albicans cells counted microscopically were added. The sensitivity limit was 20 cells per 10 ml blood. Only 5 μl of the DNA (total volume after purification 75 μl) was used for this amplification. Appropriate Experiments with 200 and 2000 cells per 10 ml blood gave significantly higher hybridization signals. The melting temperature in the FRET mode was 63 ° C. DNA from a variety of different puddle species was analyzed in this way. However, both in SybrGreen mode and in FRET / Hybprobe mode, only amplifications could be detected in samples, the human pathogenic puddle of the species C. albicans, C. guiUiermondii, C. maltosa, C. parapsüosis, C. tropicalis, C. viswanatii, C. glabrata, C. crusei, A. fumigatus, A.niger, A. nidulans, A. flavus, A. terreus, A. awamori, A. oryzae, A.parasiticus, A. soentaltee, A. tamarii A. sojae , A. wentii, Arestrictus, A. ochraceus, A. clavatus or A. candidus.

Claims

Expectations

1. Multiplex amplification reaction for the detection of clinically relevant purging infections of the genera Candida and Aspergillus, in which a region of the 18s RNA gene of the pathogen is amplified as the target, characterized in that only sequences of pathogenic Candida or Aspergillus species are amplified.

2. Multiplex ampHfikationsreaction for the detection of clinically relevant infections of the genus Candida and Aspergülus, in which a region of the 18s RNA gene of the pathogen is amplified as a target, characterized in that sequences of the Candida and Aspergillus species C. albicans, C. guiUiermondii, C maltosa, C. parapsüosis, C. tropicalis, C. viswanatii, C. glabrata, C. crusei, A. fumigatus, A.niger, A. nidulans, A. flavus, A. terreus, A. awamori, A. oryzae , A. parasiticus, A. soente, A. tamarii A. sojae, A. wentii, A. restrictus, A. ochraceus, A. clavatus and A. candidus. be amplified.

3. Multiplex amplification reaction for the detection of clinically relevant pure infections of the genus Candida, in which a region of the 18s RNA gene of the pathogen is amplified as the target, characterized in that sequences of the Candida species C. albicans, C. guüliermondn, C. maltosa, C parapsüosis, C. tropicalis, C. viswanatii, C. glabrata and C. crusei. be amplified.

4. The method according to claim 1-3, characterized in that exactly one upstream primer is used, the sequence of which is identical to a sequence of the 18 sRNA gene which is identical in the species to be detected.

5. Primer with a length of 13-25 nucleotides, the sequence of which is a continuous partial sequence of a sequence according to Seq. ID. No. 1 represents.

6. Primer with a length of 13-25 nucleotides, the sequence of which is a continuous partial sequence of a sequence according to Seq. ID. No. 2 represents.

7. Primer with a length of 13-25 nucleotides, the sequence of which is a continuous partial sequence of a sequence according to Seq. ID. No. 3 represents.

8. Primer with a length of 13-25 nucleotides, the sequence of which is a continuous partial sequence of a sequence according to Seq. ID. No. 4 represents.

9. Primer with a length of 13-25 nucleotides, the sequence of which is a continuous sequence of parts of a sequence according to Seq. ID. No. 5 figure.

10. The method according to claim 4, characterized in that one, more or all of the primers according to claims 4-8 are used.

11. The method according to claim 1-4 or 10, characterized in that the product of the amplification reaction is additionally detected by hybridization.

12. The method according to claim 11, characterized in that the hybridization probes used are identical to or complementary to a partial sequence other than 18s RNA sequences which were amplified during the amplification reaction.

13. Probe with a length of 20 to 100 and preferably 25-35 nucleotides, which has a continuous sequence according to Seq. ID. No. 6 contains.

14. The method according to claim 12, characterized in that a probe according to claim 13 is used.

15. The method according to claim 1-4, 10-12 or 14, characterized in that the amplification product is detected by means of fluorescence detection.

16. The method according to claim 15, characterized in that the amplification product is detected by means of fluorescence resonance energy transfer.

17. The method according to claim 16, characterized in that hybridization probes according to Seq. ID. No. 7 and 8 can be used.

18. Kit containing primers with sequences according to Seq. ID. No. 1-4 or Seq. ID No. 1-5.

19. Kit according to claim 18, containing hybridization probes.