The United States of America, As Represented by the Secretary, Department of Health and Human Services

The United States of America As Represented by the Department of Health

Ericsson Gmbh

The United States of America

The Government of the United States of America, As Represented by the

The United States of America As Represented by the Secretary of Health

Centers for Disease Control and Prevention

Christine J Morrison

Errol Reiss

Timothy J Lott

Brent Lasker

Sandra Zakroff

Jong Soo Choi

Liliana Aidorevich

Hans Peter Hinrikson

Brian Holloway

Teresa Brown

Mark D Lindsley

Jong S Choi

Sanchita Das

Cheryl M Elie

Zhenyu Qin

Jong Hee Shin

Compositions and methods for detecting and/or differentiating amongorganisms, includingand, are disclosed. Exemplary methods involve screening a sample suspected of containing at least one or moresp. for the presence or absence of a nucleic acid sequence specific for each such fungal pathogen. Some disclosed methods permit the rapid and simultaneous detection and identification of several fungal pathogens (e.g., up to 100 fungi) in a single sample.

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Novel techniques for the detection ofin samples are disclosed. These techniques relate to PCR amplification and/or detection ofITS1 rDNA sequences, and the identification of particular species ofby detecting differences in the ITS1-V1, ITS-V2, ITS-V3, ITS-V4, and ITS-V5 nucleic acid sequences of. The highly variable regions of the ITS1 rDNA sequences are particularly useful in distinguishing, for example,, and. In particular embodiments, the sequence differences are also able to distinguish among variants of particular species, such asCBS 119.5A,strain NRRL 1932,strain NRRL 250,strain NRRL 4768,strain CUHI,strain CUH2,strain CUH7, andstrain CUH8.

Methods of detecting a dimorphic fungus, including differentiating a dimorphic fungus from other fungi are disclosed. A sample suspected of containing a nucleic acid of a fungus, such as an internal transcribed spacer-2 (ITS2) nucleic acid sequence of a dimorphic fungal rDNA, is screened for the presence or absence of that nucleic acid. The presence of the nucleic acid indicates the sample was contacted by the fungus. Determining whether the nucleic acid sequence is present in the sample can be accomplished by detecting hybridization between a dimorphic probe, species-specific probe, and/or microbe-specific probe and a nucleic acid sequence corresponding to the ITS2 region of fungal rDNA. Kits and arrays for carrying out these methods also are disclosed.

Methods for the differentiation and identification of medically important endemic fungi

Nucleic acids for detectingspecies and other filamentous fungi are provided. Unique internal transcribed space 2 coding regions permit the development of nucleic acid probes specific for five different species of, three species of, four species of, two species of, five species of, one species of, as well as probes for, and. Methods are disclosed for the species-specific detection and diagnosis of infection byorin a subject. Furthermore, genus-specific probes are also provided forand, in addition to an all-fungus nucleic acid probe.

Nucleic acids for detecting fusarium species

Nucleic acids for detecting Aspergillus species and other filamentous fungi are provided. Unique internal transcribed space 2 coding regions permit the development of nucleic acid probes specific for five different species of Aspergillus, three species of Fusarium, four species of Mucor, two species of Penecillium, five species of Rhizopus, one species of Rhizomucor, as well as probes for , and . Methods are disclosed for the species-specific detection and diagnosis of infection by Aspergillus, Fusarium, Mucor, Penecillium, Rhizomucor, , Cunninghaemella, Pseudallescheria or Sporthrix in a subject. Furthermore, genus-specific probes are also provided for Aspergillus, Fusarium and Mucor, in addition to an all-fungus nucleic acid probe.

Nucleic acids for detecting Aspergillus species and other filamentous fungi

The present invention provides apparatus and methods in which variable delay is provided for channel tracking. In particular, a demodulator having both a hard output (or symbol estimate) and a soft output (or indication of the quality of the symbol estimate) is coupled to a channel tracker. The channel tracker provides a channel estimate used by the demodulator, in part, to correct for fading. The channel tracker updates the channel estimate based on the symbol estimates from the demodulator and the received signal. However, when the soft output indicates a low confidence in the symbol estimate, the symbol estimate is not used to update the channel estimate. This is accomplished by providing a variable delay to the channel estimate calculation performed by the channel tracker depending on the number of consecutive suspect symbol estimates. For example, a single suspect estimate generates a delay of one causing the most recent estimate to not be used, two consecutive suspect estimate generates a delay of two causing the two most recent estimates to not be used and so on. Accordingly, the channel tracker of the present invention utilizes only the most reliable data available locally to generate a channel estimate for use by the demodulator.

Apparatus and methods for variable delay channel tracking

The nucleic acid sequence encoding the internal transcribed spacer 2 region of Candida, the organism causing candidiasis, for various Candida species. Nucleic acid molecules useful as probes for detecting Candida species are described. The nucleic acid molecules are useful in methods for the detection and diagnosis of Candida infection in a sample or subject.

Nucleic acid probes for detecting Candida species

The present invention provides a rapid method for identifying species of Candida. Identification is through the use of a non-conserved regions of the ITS2 region flanked by highly conserved functional domains. Detection of members of the Candida and Aspergillus genera is enhanced by the detection of genus-specific regions of the 5.8S rRNA gene. The present invention provides isolated nucleic acids that selectively hybridize to the fungal genus-specific 5.8S rRNA region, and to the species-specific ITS2 region. The invention provides for nucleic acid sequences for use as selective probes for fungal genus-specific probes and as Candida species-specific probes. The present invention provides methods for the use of the Candida species-specific probes that allow the detection and identification of the individual species in biological samples.

Methods and compositions for the detection of Candida spp.

Provided is an isolated double-stranded nucleic acid consisting essentially of the nucleotide sequences defined in the Sequence Listing by SEQ ID Nos:5-9. These are the ITS2 sequences for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. A method of diagnosing systemic candidiasis in a subject is also provided. The method comprises the steps of: (a) collecting blood from the subject into tubes containing detergent, polypropylene glycol, sodium poyantholesulfonate, and sodium ethylene diamine tetraacetic acid; (b) lysing Candida cells using ZYMOLYASE.RTM.-100T.TM. enzyme mixture with agitation; (c) extracting and precipitating the DNA from the lysed cells; (d) amplifying the precipitated DNA using universal fungal primer pairs derived from the internal transcribed spacer regions of the Candida ribosomal DNA; and (e) detecting amplified DNA from Candida by hybridizing the amplified DNA with a probe that selectively hybridizes with Candida DNA, the presence of amplified DNA indicating systemic candidiasis.

Nucleic acid probes for candida parapsilosis and methods for detecting

Provided is an isolated double-stranded nucleic acid consisting essentially of the nucleotide sequences defined in the Sequence Listing by SEQ ID Nos: 5-9. These are the ITS2 sequences for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. A method of diagnosing systemic candidiasis in a subject is also provided. The method comprises the steps of: (a) collecting blood from the subject into tubes containing detergent, polypropylene glycol, sodium poyantholesulfonate, and sodium ethylene diamine tetraacetic acid; (b) lysing Candida cells using ZYMOLYASE.RTM.-100T with agitation; (c) extracting and precipitating the DNA from the lysed cells; (d) amplifying the precipitated DNA using universal fungal primer pairs derived from the internal transcribed spacer regions of the Candida ribosomal DNA; and (e) detecting amplified DNA from Candida by hybridizing the amplified DNA with a probe that selectively hybridizes with Candida DNA, the presence of amplified DNA indicating systemic candidiasis.

Nucleic acid sequences and methods for detecting candida tropicalis in

Provided is an isolated double-stranded nucleic acid consisting essentially of the nucleotide sequences defined in the Sequence Listing by SEQ ID Nos:5-9. These are the ITS2 sequences for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. A method of diagnosing systemic candidiasis in a subject is also provided. The method comprises the steps of: (a) collecting blood from the subject into tubes containing detergent, polypropylene glycol, sodium poyantholesulfonate, and sodium ethylene diamine tetraacetic acid; (b) lysing Candida cells using ZYMOLYASE.RTM.-100T with agitation; (c) extracting and precipitating the DNA from the lysed cells; (d) amplifying the precipitated DNA using universal fungal primer pairs derived from the internal transcribed spacer regions of the Candida ribosomal DNA; and (e) detecting amplified DNA from Candida by hybridizing the amplified DNA with a probe that selectively hybridizes with Candida DNA, the presence of amplified DNA indicating systemic candidiasis.

Nucleic acid probes and methods for detecting Candida krusei cells in

Provided is an isolated double-stranded nucleic acid consisting essentially of the nucleotide sequences defined in the Sequence Listing by SEQ ID NOs: 5-9. These are the ITS2 sequences for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. A method of diagnosing systemic candidiasis in a subject is also provided. The method comprises the steps of: (a) collecting blood from the subject into tubes containing detergent, polypropylene glycol, sodium poyantholesulfonate, and sodium ethylene diamine tetraacetic acid; (b) lysing Candida cells using ZYMOLYASE.RTM.-100T with agitation; (c) extracting and precipitating the DNA from the lysed cells; (d) amplifying the precipitated DNA using universal fungal primer pairs derived from the internal transcribed spacer regions of the Candida ribosomal DNA; and (e) detecting amplified DNA from Candida by hybridizing the amplified DNA with a probe that selectively hybridizes with Candida DNA, the presence of amplified DNA indicating systemic candidiasis.

Nucleic acid probes and methods for detecting Candida glabrata DNA in

Provided is an isolated double-stranded nucleic acid consisting essentially of the nucleotide sequence defined in the Sequence Listing by SEQ ID NO:5. This is the C. albicans ITS2 sequence and includes a nucleic acid comprising a nucleotide sequence that is specific for Candida albicans. Further examples of an isolated double stranded nucleic acid of the present invention consist essentially of the nucleotide sequences defined in the Sequence Listing by SEQ ID NOs:6-9. These are the ITS2 sequences for C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. A method of diagnosing systemic candidiasis in a subject is also provided. The method comprises the steps of: (a) collecting blood from the subject into tubes containing detergent, polypropylene glycol, sodium polyanetholesulfonate, and sodium ethylene diamine.RTM. tetraacetic acid; (b) lysing Candida cells using ZYMOLYASE.RTM.-100T with agitation; (c) extracting and precipitating the DNA from the lysed cells; (d) amplifying the precipitated DNA using universal fungal primer pairs derived from the internal transcribed spacer regions of the Candida ribosomal DNA; and (e) detecting amplified DNA from Candida by hybridizing the amplified DNA with a probe that selectively hybridizes with Candida DNA, the presence of amplified DNA indicating systemic candidiasis.

Growing community of inventors

Decatur, GA, United States of America

Christine J Morrison