Method for the simultaneous detection of at least one fungus, in particular mold and yeast, in a sample comprising the steps of: a) providing a sample suspected of containing at least one fungus, b) contacting said sample with at least one primer pair derived from SEQ ID No. LNA oligos can bind to DNA in a sequence- specific manner so that binding does not interfere with plasmid conformation or gene expression. 15) and 5 ' -CGCGT- TACTTGGGAGTGTAGC-3 ' (SEQ ID No. The reverse primer of the primer pair is preferably selected from the group consisting of 5 ' -AAGTTCTTTTCATCTTTCCWTCACWGT-S ' (SEQ ID No. 24) as nucleic acid probe is contacted with the sample. The present invention is further illustrated by the following figures and examples. 1 shows a multiple alignment of fungal 28S r RNA gene sequences with homologous human DNA regions. Method according to claim 1 or 2, characterized in that the at least one fungus is selected from the group consisting of Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus candidus, Aspergillus clavatus, Aspergillus ochraceus, Aspergillus peni- cillioides, Aspergillus ustus, Aspergillus versicolor, Candida albicans, Candida cylindracea, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida incon- spicua, Candida kefyr, Candida krusei, Candida lambica, Candida lusitaniae, Candida membranaefaciens, Candida norvegensis, Candida parapsilosis, Candida pelliculosa, Candida rugosa, Candida sake, Candida tropicalis, Candida utilis, Candida allociferii, Candida colliculosa, Candida lipolytica, Candida zeylanoides, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Fusarium verticillioides, Acremonium strictum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon moniliiforme, Trichosporon asteroides, Trichosporon ovoides, Trichosporon mucoides, Mucor circinelloides, Mucor hiemalis, Mu- cor mucedo, Mucor racemosus, Mucor ramosissimus, Mucor amphi- biorura, Mucor rouxii, Rhizopus oryzae, Rhizopus azygosporus, Rhizopus microsporus, Rhizopus stolonifer, Absidia corymbifera, Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus neo- formans, Scedosporium apiospermum, Scedosporium proliderans, Al- ternaria alternata, Histoplasma capsulatum, Penicillium chryso- genum, Cladosporium cladosporioides, Cladosporium oxysporum, Blastoschizomyces capitatus, Malassezia furfur, Malassezia sym- podialis, Saccharomyces cerevisiae, Rhodotorula xnucilaginosa, Apophysomyces elegans, Basidiobolus ranarum, Cokeromyces re- curvatus, Cunninghamella bertholletiae, Mortierella wolfii, Sak- senaea vasiformis, Geotrichum candidum and combinations thereof.4. LNA is a bicyclic nucleic acid where a ribonucleos- ide is linked between the 2 ' -oxygen and the 4 '-carbon atoms with a methylene unit (see e.g. Locked nucleic acids (LNA) are a class of conformationally restricted oligonucleotide analogues. In particular one or both of the nucleotides in parenthesis of the reverse primers 5 '-CT (T) TYCAAAGTGCTTTTCA (T) C-3 (SEQ ID NO. In order to avoid cross reactions with human DNA it turned out that especially those regions of the primers may suitably be modified with LNA bases. The use of these primers and this probe allows for the specific detection of fungi of the genus Candida, if present, in a sample. Method according to any one of claims 1 to 9, characterized in that the forward and the reverse primer of the at least one primer pair consists of 15 to 40, preferably of 16 to 35, more preferably of 17 to 30, nucleotides.12. - -- optionally at least one nucleic acid probe, in particular a labelled nucleic acid probe, which sequence is derived from SEQ ID No. A method of choice can be polymerase chain reaction (PCR) . Of course the clinical relevance of a single fungus varies from region to region. The size of the primers used in a method according to the present invention can vary. For instance, 5'-ACT(T)GT(G)CG(C)TA(T)CG-3' (SEQ ID No. 26), 5'-CT (T) TYCAAAGTGCTTTTCA (T) C-3' (SEQ ID No.
24) as nucleic acid probe is contacted with the sample.18. 31) and optionally a positive control specific nucleic acid probe δ'-TTTTTATGTGTCCGCCACCATCTGGATC-S' (SEQ ID NO. The observation that early initiation of antifungal treatment significantly improves the outcome in neutropenic patients with IFI has provided the basis for empirical rather than evidence-based antifungal treatment of patients with febrile neutropenia (Denning, 1998). In order to apply to said patients an appropriate therapy it is of major importance to unequivocally determine the presence of a fungus in said patient. According to a preferred embodiment of the present invention the amplified nucleic acid product is detected by gel electrophoresis, Southern-blot, photometry, chromatography, colori- metry, fluorography, chemiluminescence, autoradiography, detection by specific antibody and combinations thereof. It is an object of the present invention to provide reliable, reproducible, highly specific, sensitive and robust methods and means for the detection of fungi, especially fungi of clinical relevance, in a sample. 6, SEQ ID No, 7 or combinations thereof, wherein the primer pair consists of one forward and one reverse primer, c) subjecting the sample contacted with said at least one primer pair to a nucleic acid amplification technique, and d) optionally determining the presence of the at least one fungus in said sample by detecting a nucleic acid amplification product . The use of primers derived from a sequence alignment of more than one nucleotide sequence allows for the "simultaneous detection" of at least one fungus in a sample because these primers are able to bind to the DNA of a number of fungi. In the US 5, 919, 617 a method for the identification of fungal pathogens using conserved regions of saccharopine dehydrogenase of Candida albicans is described. In the nucleic acid sequences provided herein "A" stands for adenosine, "T" for thymidine, "G" for guanosine, "C" for cytosine, "N" for adenosine, thymidine, guanosine or cytosine, "V" for adenosine, guanosine or cytosine, "D" for adenosine, thymidine or guanosine, "B" for thymidine, guanosine or cytosine, "H" for adenosine, thymidine or cytosine, "W" for adenosine or thymidine, "S" for guanosine or cytosine, "K" for thymidine or guanosine, "M" for adenosine or cytosine, "Y" for thymidine or cytosine and "R" for adenosine or guanosine. (2005) Bone Marrow Transplant 9-95; (6) Maaroufi, Y., et al. The method according to the present invention may also be used to detect at least one fungus in a sample e.g. According to the present invention "derived from" means that the primers or probes, which may comprise at least 10, preferably at least 15, nucleotides, are directly deduced from the nucleotide sequences (alignments or individual sequences) disclosed herein (SEQ ID No. 7 which is located between the forward and reverse primer. According to a preferred embodiment of the present invention, the nucleic acid amplification technique is a polymerase chain reaction technique, which may be selected from the group consisting of real-time PCR, preferably Taq Man PCR, quantitative PCR, nested PCR, asymmetric PCR, multiplex PCR, inverse PCR, rapid PCR and combinations thereof. homogen- isation and/or fractionation of the sample, purification or precipitation of the nucleic acid) or used directly for the nucleic acid amplification. When analyzing solid material the sample may be obtained by washing said material with a fungus-free solution. Depending on the dye or dye/quencher combination used, the amplified product may be detected during or after the application of the nucleic acid amplification technique.
Ung dating Gribskov
7, _ Detection of fungi The present invention relates to a method for the simultaneous detection of fungi in a sample. (1988) PNAS USA 85: 2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) Nucleic Acids Res., 12, 387-395), BLASTP, BLASTN, FASTA (Atschul, S. Default parameters for the GAP program can include : (1) a unary comparison matrix (containing a value of 1 for identities and for non-identities) and the weighted comparison matrix of Gribskov et al . , ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. In order to unequivocally determine the presence of a fungus in a sample, a nucleic acid probe which is able to bind to the nucleic acid amplification product is added to the sample prior to or after the application of the nucleic acid amplification technique. They have the potential to not only substantially increase the sensitivity of the PCR, but also to provide the means to monitor re- sponse to therapy by measuring fungal burden at any given point in time. These primers can be obtained by simple chemical synthesis (e.g. Whether any two nucleic acid molecules have nucleotide sequences that are "identical" to a certain degree ("% identity") can be determined using known computer algorithms such as the "FAST A" program, using for example, the default parameters as in Pearson et al. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter one of the two sequences. Advanced technologies, such as real-time quantitative PCR (RQ-PCR) eliminate the post-amplification handling steps. Method according to any one of claims 1 to 12, characterized in that the forward primer of the primer pair is selected from the group consisting of 5 ' -GGGTGGTAAATTCCATCTAARGCTAA-S ' (SEQ ID No. 9), 5 ' -GG- GTGGTAAATTYCATCTAARGCTAA-3 ' (SEQ ID No. Table 1: Ribosomal r DNA gene targets for PCR-based fungal detection Gene target Ref. zeylanoides, Lodderomyces elongisporus, P.guilliermondii Abbreviations: SSU, small subunit; LSU, large subunit; ITS, internal transcribed spacer; RQ-PCR, real-time quantitative PCR; References: (1) Chen, Y. It turned out that especially primers with 15 to 40 nucleotides are valuable. For instance, fungi of the genera Aspergillus and Candida may be detected when the primers SEQ ID. 10 and 23, option- - - ally in combination with the probe SEQ ID No. Fun- gi, which are members of various genera, may be detected, e.g., if SEQ ID No. 20), 5 ' -A(C) TT (G) T (T) C (G) (C) TA (T) CG-3 ' (SEQ ID No. 33), 5'-TAC(T)T(G)TK(C) (G)CT(A)TCGGT-3' (SEQ ID No. In particular, it is advantageous to substitute nucleotides with LNA nucleotides in those regions of the primers which are, when aligned to human DNA sequences, differing from the human DNA. Method according to any one of claims 1 to 11, characterized in that the primers of the at least one primer pair and/or the at least one nucleic acid probe comprise at least one LNA (locked nucleic acid) nucleotide.13. Recently, a variety of PCR methods based on the detection of fungal DNA have been published (see Table 1) . According to another preferred embodiment of the present invention the sample of step a) is further contacted with at least one nucleic acid probe which sequence is derived from SEQ ID No. When sequence specific primers are designed, it has to be considered that the primer length influences the specificity of the primer as well as the melting temperature of the respective primer-DNA complex. The nucleic acid probe is selected, of course, so that it can specifically bind to a nucleic acid fragment amplified with a primer pair. 19), 5 ' -CTCT (T) TTCAAAGTW- CTTT TCA(Y)C (SEQ ID No. 35), wherein the nucleotides in parenthesis may be all or in part substituted with corresponding LNA nucleotides, are preferably employed in a method or kit according to the present invention. 7 or combinations thereof and which sequence is located between the forward and the reverse primer derived from SEQ ID No. RQ-PCR ITSl, 5.8S r DNA, ITS2 (9) Panfungal Fragment analysis (3) Panfungal Southern blot (3) Panfungal Nested PCR (1) Yeasts Fragment analysis (2) Yeasts Fragment analysis (7) Mold RQ-PCR (3) Candida spp. Southern blot (4) Candida spp., RQ-PCR Cryptococcus neoformans (6) C. albicans, C.glabrata, RQ-PCR C.krusei, C.parapsilosis, C.kefyr, C. albicans, C.glabrata, Southern blot C.krusei, C.parapsilosis, C. - - According to a preferred embodiment of the present invention, the primers of the at least one primer pair and/or the at least one nucleic acid probe comprise at least one LNA (locked nucleic acid) nucleotide. At least one fungus of the genus Candida is preferably detected when 5'-GGGTGGTAAATTCCATCTAARGCTAA-S' (SEQ ID NO.
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