EP1379687A2 - Detection d'especes rares de candida - Google Patents

Detection d'especes rares de candida

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Publication number
EP1379687A2
EP1379687A2 EP01992792A EP01992792A EP1379687A2 EP 1379687 A2 EP1379687 A2 EP 1379687A2 EP 01992792 A EP01992792 A EP 01992792A EP 01992792 A EP01992792 A EP 01992792A EP 1379687 A2 EP1379687 A2 EP 1379687A2
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
binds
nucleic acid
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01992792A
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German (de)
English (en)
Inventor
Hermann Einsele
Jürgen LÖFFLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eberhard Karls Universitaet Tuebingen
Original Assignee
Eberhard Karls Universitaet Tuebingen
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Filing date
Publication date
Application filed by Eberhard Karls Universitaet Tuebingen filed Critical Eberhard Karls Universitaet Tuebingen
Publication of EP1379687A2 publication Critical patent/EP1379687A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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

Definitions

  • the present invention relates to a nucleic acid molecule, a kit containing the nucleic acid molecule, the use of the nucleic acid molecule for. Detection of fungi or for determining the sequence of ribosomal fungal genes, a method for the detection of fungi in clinical material and a kit for carrying out this method.
  • Nucleic acid molecules which are used for the detection of fungi or for the sequence determination of ribosomal fungal genes, as well as methods for the detection of fungi in clinical material are known from various publications.
  • the problem of fungal infections has grown considerably over the past 20 years. This is primarily due to the increase in patients with weakened immune systems, intensive immunosuppressive chemotherapy, the increasing use of broad-spectrum antibiotics and central venous catheters.
  • Important mycoses, ie infectious diseases caused by fungi are, for example, Candida mycosis, Aspergillus mycosis or Mucor mycosis.
  • Candida mycosis is a life-threatening infection in immunocompressed hosts, e.g. Bone marrow or organ transplant recipients, in patients with extensive chemotherapy and in AIDS patients.
  • systemic Candida infections are observed in patients after extensive surgical interventions or after burns, with intensive antibiotic therapy, indwelling catheters, patients with diabetes mellitus and in older patients; see Wenzel, R.P., 1995, "Nosocomial candidemia: risk factors and attributable mortality", Clin. Infect. Dis. 20: 1531-1534 and Dean, D.A., Burckard, K.W., 1996, "Fungal infection in surgical patients", Am. J. Surg. 171: 374-382.
  • Aspergillosis is an infectious disease caused by the genus Aspergillus, which mainly leads to diseases of the respiratory system, but also of the skin and other organs.
  • PCR polymerase chain reaction
  • nucleic acids are known from DE 195 30 332 C2, DE 195 30 33 C2 and DE 195 30 336 C2, by means of which PCR-based detection of fungal DNA of different fungal species is possible.
  • two nucleic acid molecules are described which have the sequences SEQ ID Nos.
  • DE 196 35 347 C1 discloses the nucleotide sequences of four further hybridization probes with which the species-specific detection of the fungal DNA of Pneumocystis karnii, Malassezia for (SEQ ID No. 18 from the sequence listing enclosed here), Tric osporum cuaneum / Tri ⁇ osporum capitatum (SEQ ID No. 19 from the sequence listing enclosed here) as well as from Fusarium solani / Fusarium oxysporum.
  • Candida albicans Candida tropicalis
  • Candida parapsilosis Candida glabrata
  • Candida krusei Candida guillermondii
  • Candida kefyr Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans, Aspergillus versicolor.
  • the object of the present invention is therefore to provide a nucleic acid molecule of the type mentioned at the outset with which the disadvantages mentioned above are avoided.
  • nucleic acid molecule mentioned at the outset by having one of the following nucleotide sequences:
  • the object underlying the invention is hereby completely achieved.
  • the inventors have recognized that the above-mentioned nucleotide sequences, on the one hand, detect fungal DNA from the entire field of fungi, and on the other hand, species-specific detection of fungal DNA, in particular of those species that have so far been relatively insignificant but increasingly important, such as, for example, succeeds certain Candida species.
  • the underlying problem is solved according to the invention not only by a nucleic acid molecule with one of the sequences SEQ ID No. 1 to 4 or SEQ ID No. 7 to SEQ ID No. 17 from the enclosed sequence listing, but also by such a nucleic acid molecule which is attached to the same Binds sequences to which the nucleic acid molecule also binds with one of the sequences SEQ ID No. 1 to 4 or SEQ ID No. 7 to SEQ ID No. 17.
  • nucleic acid molecules are hereby recorded in which the sequences listed in the sequence listing are part of a longer sequence. Even if individual nucleotide exchanges are carried out for the sequences listed in the sequence listing, these molecules usually retain. their highly selective affinity for fungal DNA. Consequently, a nucleic acid molecule characterized in this way is also the subject of the present invention.
  • a nucleic acid molecule with one of the sequences SEQ ID Nos. 1 to 10 is particularly suitable for the detection of a general one Fungal infection.
  • This nucleic acid molecule binds to the 18S rRNA gene, which is highly conserved throughout the mushroom kingdom.
  • a database analysis in the Blast Search Program of the National Center for Biotechnology Information showed the comparative sequence analysis of the sequences SEQ ID No. 1 to 10 homologies to over 500 different fungal species. The inventors conclude from this that the nucleic acid molecule mentioned above can be used to detect fungal DNA from the entire fungal realm.
  • this ensures that even previously insignificant or unusual mushroom species are recorded. Due to the binding properties of the above-mentioned nucleic acid molecule even under stringent conditions, this can be e.g. use as a polymerase chain reaction (PCR) primer for the amplification of small amounts of fungal DNA or for sequencing previously unknown 18S rRNA genes of the fungi.
  • PCR polymerase chain reaction
  • a nucleic acid molecule with one of the nucleotide sequences SEQ ID No. 1 to 10 is therefore particularly suitable for a quick and reliable assessment of a possible general fungal infection on the basis of clinical material, for example blood or tissue samples, regardless of the fungal species causing it.
  • nucleic acid molecule with one of the nucleotide sequences SEQ ID No. 11 to SEQ ID No. 19 from the enclosed sequence listing is particularly suitable for use as a hybridization probe, for example in the context of a species-specific analysis for the presence of rare Candida species. Based on the above, it is particularly preferred to use the nucleic acid molecule according to the invention for the detection of fungi, preferably in clinical material.
  • the detection of fungi takes place via the amplification of a fungus DNA segment by means of polymerase chain reaction (PCR), in which the primers of a PCR primer pair have nucleotide sequences in a combination which is selected from the group: SEQ ID Nos. 1 and 2; SEQ ID Nos. 3 and 4; SEQ ID Nos. 7 and 8; SEQ ID Nos. 9 and 10 from the enclosed sequence listing; two sequences that bind to those sequences to which SEQ ID No. 1 and 2 or SEQ ID 3 and 4 or SEQ ID No. 7 and 8 or SEQ ID No. 9 and 10 also bind.
  • PCR polymerase chain reaction
  • segments of fungal DNA which is often only present in small amounts in clinical material are enriched to such an extent that they can be obtained using simple methods, e.g. Ethidium bromide staining after agarose gel electrophoresis can be detected.
  • nucleic acid molecules with the nucleotide sequences SEQ ID Nos. 1 to 10 listed in the sequence listing were successfully tested by the inventors as PCR primers, ie their use for the detection of fungi provides reliable results for each of these nucleic acid molecules in a highly specific and sensitive manner.
  • PCR primers When used in a PCR, there is no co-amplification of human nucleic acids or of any bacterial or viral nucleic acids that may be present.
  • PCR primer pair No. 1 SEQ ID No. 1 and SEQ ID No. 2;
  • PCR primer pair No. 2 SEQ ID No. 3 and SEQ ID No. 4;
  • PCR primer pair No. 3 SEQ ID No. 5 and SEQ ID No. 6;
  • PCR primer pair No. 4 SEQ ID No. 7 and SEQ ID No. 8 or
  • PCR primer pair No. 5 SEQ ID No. 9 and SEQ ID No. 10.
  • nucleic acid molecules with nucleotide sequences which bind to nucleotide sequences to which one of the nucleotide sequences SEQ ID No. 1 to 10 binds can also be used here as a PCR primer pair in a corresponding combination.
  • particular consideration is given to those nucleic acid molecules in which one of the sequences listed in the sequence listing is part of a longer sequence, or in which point mutations and / or deletions, substitutions, additions of nucleotides, etc. have occurred in isolated cases compared to the sequences given in the sequence listing to have.
  • nucleic acid molecules as PCR primers in the combination given above leads to optimal amplicon sizes and thus ensures the reliable detection of existing fungal DNA.
  • a specific / or genus-specific analysis of the fungal DNA is carried out by hybridization with a nucleic acid molecule which has one of the nucleotide sequences SEQ ID No. 11 to SEQ ID No. 17 from the enclosed sequence listing or a sequence which binds to such a sequence to which one of the sequences SEQ ID No. 11 to SEQ ID No. 17 also binds.
  • the fungal DNA is applied as a mask using a perforated plate a nylon carrier membrane is applied and the membrane is then incubated with the appropriately labeled hybridization probes. After subsequent washing steps under stringent conditions, hybridization to the fungal DNA can be recognized on the basis of an optically detectable signal, visual quantification being facilitated by the sample concentration in a small area.
  • a particularly high sensitivity of the detection method can be achieved, ie up to a detection limit of 100 fg fungal DNA in clinical material.
  • the detection of Candida DNA is carried out with a nucleic acid molecule which has the nucleotide sequence SEQ ID No. 11 from the enclosed sequence listing or a sequence that binds to a sequence to which the sequence SEQ ID No. 11 binds.
  • a general Candida infection for example an invasive candidiasis
  • a general Candida infection is usually diagnosed in the clinic using methods which involve the cultivation of fungal cells. It is particularly problematic here that a result of such a cultivation approach can only be achieved after several days. After this time, it is often too late for effective treatment for Candida infection.
  • Another problem is that Candida As a commensal of human skin, contaminations often lead to cultivation approaches and false-positive results. Even more problematic is the fact that in up to 50 percent of systemic candidiasis cases examined by autopsy, the underlying blood cultures were negative, making this procedure of no diagnostic value. This has led to the widespread opinion among clinicians that invasive Candida infections can generally only be diagnosed via autopsies.
  • the above-mentioned preferred variant of the invention also provides a remedy here, since no cross-reaction of the genus-specific Candida hybridization probe with human nucleic acid is observed. Even the use of the two nucleic acid molecules described in DE 195 30 332 C2 as PCR primers does not enable the detection of a general Candida infection, since DNA of the genera Aspergillus is also amplified by these primers. Furthermore, only five different Candida species have been shown to be detected. With the The genus-specific Candida hybridization probe according to the invention thus enables for the first time an early and reliable detection of a general Candida infection.
  • the invention relates to the use of a nucleic acid molecule as a hybridization probe which has the following nucleotide sequence:
  • nucleic acid molecule for the detection of one of the abovementioned species which has a sequence which binds to a sequence to which one of the sequences SEQ ID NO. 11 to 17 binds from the sequence listing. It is further preferred to use a nucleic acid molecule for sequence determination of ribosomal fungal genes which has one of the following nucleotide sequences:
  • the specified nucleic acid molecules can e.g. the nucleotide sequence of previously unknown ribosomal fungal genes can be elucidated as a PCR primer in a combination also given. This is equally due to the ability of the indicated molecules to bind to highly conserved sections of all 18S rRNA fungal genes.
  • the present invention also relates to a method for the detection of fungi in clinical material, comprising the steps:
  • nucleic acid molecule which has one of the following nucleotide sequences: SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 7 to SEQ ID No. 17 from the enclosed sequence listing or a sequence which binds to such a sequence to which one of the sequences SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 7 to SEQ ID No. 17 also binds. Due to the favorable properties of the nucleic acid molecule claimed, such a method can be used to reliably detect and present fungi on the basis of their DNA from a large number of different clinical materials, for example whole blood or tissue samples, for example as a result of a fungal infection.
  • the invention further relates to a kit which contains a nucleic acid molecule which has one of the following nucleotide sequences: SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 7 to SEQ ID No. 17 from the enclosed sequence listing or a sequence which binds to a sequence to which one of the sequences SEQ ID No. 1 to SEQ ID No. 4 or SEQ ID No. 7 to SEQ ID No. 17 also binds. Furthermore, the present invention relates to a kit for performing the method according to the invention.
  • kits has the advantage that possible errors in the implementation of the method or the use according to the invention of the claimed nucleic acid molecule are avoided by compiling all the reagents and / or nucleic acid molecules, reaction containers or parts thereof, detailed user instructions, etc. This takes into account in particular the situation in clinics in which frequently trained personnel are entrusted with the implementation of such procedures.
  • a blood sample is taken from patients who are to be examined for a fungal infection.
  • the erythrocytes of the whole blood are lysed hypotonic with RCLB buffer (10 mM Tris [pH 7.6], 5 mM MgCl 2 , 10 mM NaCl).
  • RCLB buffer 10 mM Tris [pH 7.6], 5 mM MgCl 2 , 10 mM NaCl.
  • WCLB buffer (10 mM Tris [pH 7.6], 10 mM EDTA, 50 mM NaCl, 0.2% SDS, 200 ⁇ g Proteinase K per ml) at 65 ° C for 45 Minutes. This ensures that fungal DNA, which originates from fungal cells located in blood cells, is also detected.
  • the samples are pelleted and incubated with 50 mM NaOH at 95 ° C for 10 minutes. This is followed by neutralization with 1 M Tris [pH 7.0], followed by treatment with recombinant Lyticase (Sigma, Deissenhofen, Germany) for 45 minutes at 37 ° C., in a buffer containing 1 U Lyticase per 100 ⁇ l, 50 mM Tris [pH 7.5], 1 mM EDTA and 0.2% ß-mercaptoethanol contains to form spheroplasts. After centrifugation at 5000 g, the supernatant, which contains the human DNA and proteins, is decanted and the pellets are treated with 1 M Tris-EDTA and 10% SDS at 65 ° C.
  • Example 1 The samples obtained in Example 1 are checked for the presence of fungal DNA by means of a polymerase chain reaction (PCR) in which specifically only fungal DNA is amplified.
  • PCR polymerase chain reaction
  • the suitable PCR primers are shown in Table I.
  • the amplification reactions are carried out in a 50 ⁇ l volume which contains: 10 mM Tris [pH 9.6], 50 mM NaCl, 10 mM MgCl 2 , 200 ⁇ g bovine serum albumin / ml, 0.5 M deoxyribonucleotide trisphosphate, 100 pmol respective primer and 1.5 U Taq polymerase (Amersham, Braunschweig, Germany).
  • the extracted DNA from Example 1 (100 ng) is added and 34 cycles of repeated denaturation, primer hybridization and enzymatic chain extension are carried out in a PE 2400 thermocycler (Perkin Elmer, Dreieich, Germany).
  • the amplification program has the following profile: 30 seconds at 94 ° C, 1 minute at 62 ° C and 2 minutes at 72 ° C, followed by a cycle of terminal extension at 72 ° C for 5 minutes.
  • aliquots of a saline solution and of human fibroblast DNA are prepared and used as a negative control in the amplification reaction.
  • each amplification product is electrophoresed over a 2% agarose gel in 1 ⁇ TAE buffer (pH 8.0, 40 mM Tris acetate [pH 7.5], 2 mM sodium EDTA ) separated, followed by an ethidium bromide stain.
  • the fluorescent fungal DNA can be displayed on a screen with excitation of the ethidium bromide intercalated into the DNA.
  • the five specified PCR primer pairs are therefore suitable for the simple detection of the presence of fungi or fungal DNA, for example in patient blood, regardless of the respective fungal species.
  • a slot blot assay is carried out to further determine the fungus species or genus.
  • 10 ⁇ l aliquots of each amplicon from Example 2 are pipetted onto Hybond N + nylon membranes (Amersham, Braunschweig, Germany), onto which a slotted perforated plate is placed.
  • the table below shows the sequences of the hybridization probes labeled with digoxigenin, the corresponding melting temperatures at which half of the respective nucleic acid molecule is in a solution in the double-stranded form, the other half is in the form of a single-stranded molecule, the respective GC content, ie the Amount of guanine and cytosine residues in the molecule, and the specific washing temperature (see below).
  • Each amplicon (example 2) is hybridized with the respective probe for 20 minutes at 42 ° C. This is followed by specific washing steps for twice 7 minutes with washing buffer (100 mM sodium chloride, 10 mM sodium dihydrogen phosphate, 1 M EDTA, 1% SDS) at the probe-specific temperatures given above.
  • washing buffer 100 mM sodium chloride, 10 mM sodium dihydrogen phosphate, 1 M EDTA, 1% SDS
  • the washing temperature which is only a few degrees below the T m , and the presence of SDS in the washing buffer only allow specific hybridization reactions. or prevent unspecific attachment of probes.
  • hybrids are then incubated for 20 minutes with anti-digoxigenin antibodies, conjugated with alkaline phosphatase (Röche, Molecular Biochemicals, Mannheim, Germany) and for a further 30 minutes with nitroblue tetrazolium (75 mg / ml in dimethylformamide) and promoter-indoyl phosphate Solution (50 mg / ml in dimethylformamide, Röche Molecular Biochemicals, Mannheim, Germany).
  • alkaline phosphatase Röche, Molecular Biochemicals, Mannheim, Germany
  • a subsequent reaction enzymatic cleavage of a chromogenic substrate by the enzyme bound to the antibody forms a colored reaction product which indicates a positive reaction for the particular batch or for the respective amplificate.
  • no anti-digoxigenin antibody can be attached to the labeled one Bind probe or is washed away together with it.
  • the colored reaction product does not form, which means that the reaction is assessed as negative.
  • yeast cultures were obtained from the German Collection of Microorganisms (DMSZ, Braunschweig, Germany):
  • Candida albicans (DSM 6569), Candida glabrata (DSM 6425), Candida krusei (DSM 6128), Candida tropicalis (DSM 5991), Candida parapsilosis (DSM 5784), Candida lusitanie (DSM 70102), Candida humicola (DSM 5572), Candida pseudotropicalis (ä Candida kefyr) (ATTC 14438), Candida incosnspicua (DSM 70631), Candida solani (DSM 3315), Candida norvegensis (DSM 70760), Candida utilis (DSM 2361), Saccharomyces cerisiae (DSM 1333), Trichosporon cutaneum (DSM 70698), Malassezia for (DSM 6170), Fusarium solani (DSM 1164) and Aspergillus fumigatus (DSM 790).
  • the yeast cells were washed and resuspended in 0.9% sterile sodium chloride solution.
  • the yeast cell suspensions were titrated so that final concentrations of 10 6 to 10 1 colony-forming endings (CFU) per ml of solution were set.
  • 100 ⁇ l of the suspension containing 10 5 to 10 1 CFU were added to blood samples obtained from healthy volunteers.
  • yeast was isolated from feces, liver abscesses, sputum or blood from patients suffering from lymphoma, acute lymphoblastic leukemia or AIDS.
  • Candida probe (with the sequence SEQ ID No. 11) surprisingly detected DNA from all 15 analyzed yeast species (C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, C. . lusitaniae, C. humicola, C. pseudotropicalis (a C. kefyr), C. inconspicua, C. solani, C. nor einsis, C. utilis, S. cerivisiae, T. cutaneum and M. furfur).
  • No hybridization signal was obtained for DNA derived from Aspergillus fumigatus, Aspergillus niger, Fusarium ssp.
  • FIG. 1 The result of such a hybridization experiment or slot blot assay is shown in FIG. 1.
  • the following amounts of C. lusita ⁇ iae DNA were applied in lane 1: 100 pg (A), 10 pg (B), 1 pg (C), 100 fg (D); corresponding amounts of C. tropicalis DNA were applied in lane 2; corresponding amounts of C. glabrata DNA in lane 3; corresponding amounts of C. rusei DNA in lane 4; corresponding amounts of C. parapsilosis DNA in lane 5.
  • Aliquots of clinical isolates infected with corresponding fungal cultures were applied to points F: C. lusitaniae isolate (Fl), C. grlajrata isolate (F3), A. fumigatus isolate (F4, negative control), A. niger isolate (F5, negative control); double distilled water was applied as a further negative control (F2).
  • the species-specific hybridization probes were highly specific even under the stringent washing conditions described above. Furthermore, no cross-reactions were observed within the above-mentioned species with the hybridization probes which have one of the sequences SEQ ID Nos. 12 to 14, 16 to 19; with the exception of the hybridization probe, which has the sequence SEQ ID No. 15, which recognizes both DNA from C. norogensis and C. krusei. The latter is shown in Fig. 2. Here 10 pg DNA of the following fungal species were applied: C. humicola (1), C. solani (2), C. inconspicua (3), C. norvegensis (4), C. kefyr (5) and C. lusitaniae (6 ).
  • SEQ ID No. 12 A
  • SEQ ID No. 17 B
  • SEQ ID No. 13 C
  • SEQ ID No. 15 D
  • SEQ ID No. 16 E
  • SEQ ID No. 14 F
  • Candida species cultures were titrated (C. albicans, C. humicola, C. lusitaniae, C. inconspicua, C. nor einsis, C. pseudotropicalis (& C. kefyr), C. solani) , with concentrations of 10 5 to 10 ° CFU-adjusted. This showed a lower detection limit of at least 10 1 CFU, which corresponds to an absolute amount of 100 fg of fungal DNA.
  • This high sensitivity was found by the inventors for all special-specific probes (with the sequences SEQ ID No. 12 to SEQ ID No. 19) and for the genus-specific Candidasonde (with the sequence SEQ ID No. 11; see also FIG. 1). be documented.

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Abstract

La présente invention concerne une molécule d'acide nucléique, un ensemble contenant cette molécule d'acide nucléique, l'utilisation de cette molécule d'acide nucléique dans la détection de champignons ou dans la détermination de séquence de gènes de champignons ribosomaux, un procédé de détection de champignons dans du matériel clinique, ainsi qu'un ensemble permettant de mettre en oeuvre ce procédé.
EP01992792A 2000-10-30 2001-10-12 Detection d'especes rares de candida Withdrawn EP1379687A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10053821 2000-10-30
DE10053821A DE10053821A1 (de) 2000-10-30 2000-10-30 Detektion von seltenen Candida-Spezies
PCT/EP2001/011805 WO2002036813A2 (fr) 2000-10-30 2001-10-12 Detection d'especes rares de candida

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EP1379687A2 true EP1379687A2 (fr) 2004-01-14

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EP (1) EP1379687A2 (fr)
AU (1) AU2002245856A1 (fr)
DE (1) DE10053821A1 (fr)
WO (1) WO2002036813A2 (fr)

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WO2007035814A2 (fr) * 2005-09-20 2007-03-29 Advandx, Inc. Reactifs, methodes et trousses destines a la classification de champignons et a l'application d'un traitement antifongique
JP2007159411A (ja) * 2005-12-09 2007-06-28 Canon Inc プローブセット、プローブ固定担体及び遺伝子検査方法
GB0621864D0 (en) 2006-11-02 2006-12-13 Univ Manchester Assay for fungal infection
WO2011151473A1 (fr) * 2010-06-02 2011-12-08 2B Blackbio S.L. Composition, procédé et kit pour la détection de champignons et de levures par séquençage
WO2012050787A1 (fr) * 2010-09-29 2012-04-19 Ibis Biosciences, Inc. Extraction d'acide nucléique fongique
WO2014071946A1 (fr) * 2012-11-07 2014-05-15 Statens Serum Institut Amorces pcr de diagnostic permettant une détection exhaustive d'adnr ssu eucaryote non-humain dans des échantillons cliniques humains
US12077810B2 (en) 2016-04-14 2024-09-03 T2 Biosystems, Inc. Methods and systems for amplification in complex samples

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Publication number Priority date Publication date Assignee Title
WO1988003957A1 (fr) * 1986-11-24 1988-06-02 Gen-Probe Incorporated Sondes d'acide nucleique pour la detection et/ou la quantification d'organismes non viraux
JP3608823B2 (ja) * 1994-09-29 2005-01-12 株式会社東洋紡ジーンアナリシス 真菌の検出および真菌の菌種同定用オリゴヌクレオチド
DE19530336C2 (de) * 1995-08-17 1997-08-28 Univ Eberhard Karls Sequentielle Hybridisierung von Pilzzellen-DNA sowie Verfahren zum Nachweisen und Identifizieren von Pilzzellen in klinischem Material

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Title
See references of WO0236813A2 *

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WO2002036813A2 (fr) 2002-05-10
US20040002592A1 (en) 2004-01-01
AU2002245856A1 (en) 2002-05-15
WO2002036813A3 (fr) 2003-10-23

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