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Publication Number
US-10030278-B2
Publication Date
2018-07-24
Expiration Date
Abstract
The invention relates to a method of identifying a specific yeast species in patient tissue or body fluid. The method comprises the steps of extracting and recovering DNA of the yeast species from the patient tissue or body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the yeast species, and specifically identifying the yeast species. The invention also relates to a method of identifying a yeast mycotoxin in patient tissue or body fluid. The method comprises the steps of extracting and recovering the yeast mycotoxin from the patient tissue or body fluid, contacting the yeast mycotoxin with an antibody directed against the yeast mycotoxin, and identifying the yeast myocotoxin. Both of these methods can he used to determine if a patient is at risk for or has developed a disease state related to a yeast infection, and to develop an effective treatment regimen for the patient.
Core Innovation
A method of identifying a specific yeast species in patient tissue or body fluid is provided. The method includes extracting and recovering DNA of the yeast species from the patient tissue or body fluid, amplifying the DNA, and hybridizing a probe to the DNA, followed by specifically identifying the yeast species as Candida albicans.
The document further describes identifying other yeast species by using probe sequences consisting of specified SEQ ID NOs, with hybridization used to specifically identify the yeast species after DNA amplification. Embodiments include probe hybridization formats in which a probe is bound to fluorochrome-dyed beads, and labeling formats that involve fluorescent labeling. The amplified target can include internal transcribed spacer regions of nuclear ribosomal DNA, and the amplification and detection can be performed using real-time PCR.
In addition to yeast species identification, a method of detecting yeast-derived mycotoxins in patient tissue or body fluids is described. The method includes extracting the mycotoxin, contacting it with an antibody, and identifying the mycotoxin. The mycotoxin can be gliotoxin or patulin, with embodiments optionally including quantifying the mycotoxin, and antibody-based detection formats that may include ELISA or affinity chromatography.
The document describes diagnosis and risk determination associated with yeast infection detection, and developing a treatment regimen based on the diagnosis. Sample types and tissue formats are described, and kit and nucleic-acid composition embodiments are included. Extensive validation of real-time PCR assays is described, including specificity and cross-reactivity results and clinical urine findings for Candida albicans, Candida glabrata, Candida kruseii, and Candida tropicalis, with acceptance criteria for assay performance.
Claims Coverage
The independent claim set covers inventive features for 2 main areas: identifying a specific yeast species in patient tissue or body fluid using DNA extraction, amplification, and SEQ ID-based probe hybridization; and detecting yeast-derived mycotoxins by antibody-based identification after mycotoxin extraction, optionally including quantification.
Yeast DNA extraction from patient tissue or body fluid
Extracting and recovering DNA of the yeast species from the patient tissue or body fluid.
Amplification of recovered yeast DNA
Amplifying the DNA.
SEQ ID probe hybridization for specific yeast identification
Hybridizing a probe to the DNA to specifically identify the yeast species, wherein the probe sequence consists of SEQ ID NO: 1 or a full complement of the sequence of SEQ ID NO: 1.
Specific identification of Candida albicans
Specifically identifying the yeast species as Candida albicans.
Alternative Candida species identification using probe hybridization
Identifying a yeast species where the yeast species is selected from Candida glabrata, Candida kruseii, and Candida tropicalis.
Species probe and primer sequence set for amplification/hybridization
Using a probe, a forward primer, and a reverse primer whose sequences are SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
Amplified target as internal transcribed spacer regions
The amplified sequence is the internal transcribed spacer regions of nuclear ribosomal DNA.
Real-time PCR amplification/detection
Performing the method using real-time PCR.
Fluorochrome-dyed bead format for probe
Including a probe that is bound to a bead dyed with a fluorochrome.
Overall, the claim coverage emphasizes SEQ ID-based probe hybridization after yeast DNA extraction and amplification to specifically identify Candida albicans, with dependent refinements to cover additional Candida species, defined probe/primer sequence sets, optional targeting of internal transcribed spacer regions, real-time PCR formats, and fluorescent bead probe embodiments.
Stated Advantages
Enables specifically identifying a yeast species in patient tissue or body fluid, including Candida albicans.
Enables detecting yeast-derived mycotoxins in patient tissue or body fluids by antibody-based identification.
Includes optional quantification of the mycotoxin.
Provides extensive validation of real-time PCR assays including specificity and cross-reactivity results and clinical urine findings.
Documented Applications
Diagnosis/risk determination of yeast infection based on yeast identification and yeast-derived mycotoxin detection.
Developing a treatment regimen based on the diagnosis.
Testing patient specimens including tissue and body fluids, with clinical urine findings reported for Candida albicans, Candida glabrata, Candida kruseii, and Candida tropicalis.
Detecting yeast-derived mycotoxins such as gliotoxin and patulin in patient tissue or body fluids.
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