Ung dating Gribskov

Method according to claim 6, characterized in that the polymerase chain reaction technique is 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. Method according to any one of claims 1 to 7, characterized in that the amplified nucleic acid product is additionally tagged for detection by a technique selected from the group consisting of DNA-tagging by random-priming, DNA-tagging by nick- translation, DNA-tagging by polymerase chain reaction, oligonucleotide tailing, hybridization, tagging by kinase activity, fill-in reaction applying Klenow fragment, photobiotinylation and combinations thereof.9. 15) and 5'-CGCGTTACTTGGGAGTGTAGC- 3 ' ( SEQ ID No . Method according to any one of claims 1 to 13, characterized in that the reverse primer of the primer pair is selected from the group consisting of 5 ' -AAGTTCTTTTCATCTTTCCWTCACWGT-3 ' (SEQ ID No. Method according to any one of claims 1 to 17, characterized in that at least one fungus of the genera Aspergillus and Candida is detected when 5'-GGGTGGTAAATTYCATCTAARGCTAA-S' (SEQ ID No. Method according to any one of claims 1 to 18, characterized in that a nucleic acid as positive control and a positive control specific primer pair and optionally a positive control specific nucleic acid probe derived from said nucleic acid is added to the sample after method step a) , wherein said nucleic acid does not result in a nucleic acid amplification product when contacted and amplified with a primer pair derived from SEQ ID No. 32), when phocine herpesvirus DNA is added to the sample.22. Therefore, efforts are ongoing to develop less invasive, yet reliable, sensitive and specific diagnostic tests for IFIs to overcome the limitations of the traditional culture- based and serological fungus detection methods. The at least one fungus is preferably selected from the group consisting of Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus candidus, Aspergillus clavatus, Aspergillus ochraceus, Aspergillus penicillioides, Aspergillus ustus, Asper- gillus versicolor, Candida albicans, Candida cylindracea, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida kru- sei, Candida lambica, Candida lusitaniae, Candida membranaefa- ciens, Candida norvegensis, Candida parapsilosis, Candida pel- liculosa, Candida rugosa, Candida sake, Candida tropicalis, Candida utilis, Candida allociferii, Candida colliculosa, Candida lipolytica, Candida zeylanoides, Fusarium oxysporum, Fusarium proliferatum, Fusarium solani, Fusarium verticillioides, Acre- monium strictum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon moniliiforme, Trichosporon as- teroides, Trichosporon ovoides, Trichosporon mucoides, Mucor circinelloides, Mucor hiemalis, Mucor mucedo, Mucor racemosus, Mucor ramosissimus, Mucor amphibiorum, Mucor rouxii, Rhizopus oryzae, Rhizopus azygosporus, Rhizopus microsporus, Rhizopus stolonifer, Absidia corymbifera, Cryptococcus albidus, Crypto- coccus laurentii, Cryptococcus neoformans, Scedosporium apio- spermum, Scedosporium proliferans, Alternaria alternata, Histo- plasma capsulatum, Penicillium chrysogenum., Cladosporium cla- dosporioides, Cladosporium oxysporum, Blastoschizomyces capit- atus, Malassezia furfur, Malassezia sympodialis, Saccharomyces cerevisiae, Rhodotorula mucilaginosa, Apophysomyces elegans, Basidiobolus ranarura, Cokeromyces recurvatus, Cunninghamella bertholletiae, Mortierella wolfii, Saksenaea vasiformis, Geo- trichum candidum and combinations thereof. All these methods turned out to be suitably used in order to detect the amplification of a nucleic acid sequence. 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. 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.

ung dating Gribskov

The term "sample" includes all types of samples which may be infected by at least one fungus. 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. en dating Silkeborg 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. Furthermore, it should be also possible to quantify the amount of fungi present in a sample. 5 to 7 ) , of a large number of 28S r RNA gene sequences of fungi (see Tables 2, 4 and 5) revealed regions which are substantially - apart from some mutations - conserved regions. 2, derived from an alignment of 28S r RNA of various members of the Candida genus (see Table 2) , is shown in Fig. The consensus sequence of both Aspergillus and Candida genera results in SEQ ID No. However, it is preferred to detect the presence of at least one fungus of one or more genera with a maximum of five, preferably with a maximum of four, more preferably with a maximum of three, most preferably with a maximum of two, especially with a maximum of one, nucleic acid amplification step. The method should enable a person skilled in the art to detect a broad range of fungi or a distinct fungal genus. Sequence analysis, in particular sequence alignments (see Figs. 4 to 7 are derived from the alignments shown in Figs. 4, 5 and 6 the alignments starting from nucleotide 231 and 196 (related to A. However, it is also possible to detect in a first nucleic acid amplification reaction with a first primer pair, a group of fungi, and in at least one further nucleic acid amplification reaction at least one other group of fungi.

Ung dating Gribskov

The present invention relates to a 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... Method according to any one of claims 1 to 3, characterized in that said sample of step a) is further contacted with at least one nucleic acid probe which sequence is derived from SEQ ID No. The main mechanism for LNA oligos binding plasmid DNA is demonstrated to be by strand displacement. Consequently, the LNA modification concerning the 3' end of SEQ ID No. According to another preferred embodiment of the present invention, at least one fungus of the genus Aspergillus is detected when 5'-GGGTGGTAAATTTCATCTAAAGCTAA-S' (SEQ ID NO. The present invention relates to a 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 conserved sequences of the 28s 2RNA gene, 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.1. LNA oligos are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) clamps' for procedures such as particle mediated DNA delivery (gene gun) . 9) as forward primer, 5'-AAGTTCTTTTCATCTTTCGATCACTCT-S' (SEQ ID NO. 22) as reverse primer and optionally S'-CTTGTKCGCTATCGGTCTCTSGCCA-S' (SEQ ID NO. 7, - optionally a positive control and a positive control specific primer pair and a nucleic acid probe as defined above. 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. ung dating Gribskov-12 Method according to any one of claims 1 to 8, characterized in that the amplified nucleic acid product is detected by gel electrophoresis, Southern-blot, photometry, chromatography, col- orimetry, fluorography, chemiluminescence, autoradiography, detection by specific antibody and combinations thereof.10. 10) as forward primer, 5'-CTTTYCAAAGTGCTTTTCATC-S' (SEQ ID No. Use of a method according to any one of claims 1 to 21 for the detection of at least one fungus of the genus Aspergillus, Candida, Fusarium, Trichosporon, Mucor, Rhizopus, Acremonium, Cryptococcus, Scedosporium Alternaria, Histoplasma, Penicillium, Bipolaris, Cladosporium, Malessezia, Saccharomyces, Rhodotorula, Apophysomyces, Basidiobolus, Cokeromayces, Cunninghamella, Mor- tierella, Rhizomucor, Saksenaea and/or Geotrichum in a sample. These techniques should be replaced by rapid and sensitive procedures capable of detecting even non-culturable/non-viable cells or circulating free fungal DNA. However, the at least one fungus is most preferably selected from the group consisting of Aspergillus flavus, Aspergillus fu- migatus, Aspergillus niger, Aspergillus terreus, Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Fusarium oxysporum, Fusarium solani, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Mucor mucedo, Rhizopus oryzae, Cryptococcus albidus, Cryptococcus laurentii and Cryptococcus neoformans, which represent the most wide spread and the clinically most relevant fungi. The forward and the reverse primer of the at least one primer pair consists preferably of 15 to AO, preferably of 16 to 35, more preferably of 17 to 30, nucleotides. The primers and probes according to the present invention may comprise instead of nucleotides LNA nucleotides. in situ PCR), the isolation of a nucleic acid from a sample is not necessary.

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