Methods and compositions for detecting fungi and mycotoxins
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Publication Number
US-9103829-B2
Publication Date
2015-08-11
Expiration Date
Abstract
The invention relates to a method of identifying a specific fungal species in patient tissue or body fluid. The method comprises the steps of extracting and recovering DNA of the fungal species from the patient tissue or body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the fungal species, and specifically identifying the fungal species. The invention also relates to a method of identifying a mycotoxin in patient tissue or body fluid. The method comprises the steps of extracting and recovering the mycotoxin from the patient tissue or body fluid, contacting the mycotoxin with an antibody directed against the mycotoxin, and identifying the myocotoxin. Both of these methods can be used to determine if a patient is at risk for or has developed a disease state related to a fungal infection, and to develop an effective treatment regimen for the patient.
Core Innovation
The document describes diagnostic methods for fungal infections based on identifying specific fungal species from patient tissue or body fluid. Fungal DNA is extracted and recovered, then DNA is amplified and a fluorescently labeled isolated probe is hybridized to the DNA to specifically identify the fungal species. The probe sequence is selected from SEQ ID NO: 1, SEQ ID NO: 40, and SEQ ID NO: 67, and the method includes specifically identifying the fungal species after hybridization.
The document further specifies detection using probe and amplification formats associated with PCR and real-time PCR, and it describes amplified target regions including internal transcribed spacer regions of nuclear ribosomal DNA. It also describes fluorescent labeling formats for the probe, including embodiments where the probe is fluorescently labeled and includes binding the probe to a bead dyed with a fluorochrome. The disclosed approach emphasizes specificity and sensitivity, and a reduced time to diagnosis as supported by assay performance and patient/validation results described in the document.
In addition to fungal DNA detection, the document describes detecting mycotoxins from patient samples using mycotoxin-specific antibodies. The disclosed mycotoxin detection includes mycotoxin extraction and quantification with antibody-based assays such as ELISA and immunoaffinity, and assays using Luminex microspheres with fluorescent bead labeling. Mycotoxins explicitly described include aflatoxins (B1/B2/G1/G2), ochratoxin A, and tricothecenes, including example assay/kits such as AflaTest, OchraTest, and a Trichothecene kit.
Claims Coverage
The partial set includes two independent claims. Both independent claims share a DNA extraction, DNA amplification, and fluorescently labeled probe hybridization workflow, differing in whether the determination is diagnostic identification versus determining risk or disease state related to fungal infection.
Fluorescently labeled species-specific probe for fungal DNA identification
A method of identifying a specific fungal species in patient tissue or body fluid by extracting and recovering fungal DNA, amplifying the DNA, hybridizing an isolated probe to the DNA to specifically identify the fungal species, wherein the probe consists of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 40, and SEQ ID NO: 67, wherein the probe is fluorescently labeled, and specifically identifying the fungal species.
Assessing patient risk or fungal infection disease state using fluorescently labeled species-specific probe
A method of determining if a patient is at risk for or has developed a disease state related to a fungal infection by extracting and recovering DNA of a specific fungal species from a tissue or body fluid of the patient, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the fungal species, wherein the probe consists of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 40, and SEQ ID NO: 67, wherein the probe is fluorescently labeled, and specifically identifying the fungal species.
Across both independent claims, the core coverage is the combination of fungal DNA extraction from patient tissue/body fluid, DNA amplification, and fluorescently labeled hybridization using probes defined by SEQ ID NO: 1, SEQ ID NO: 40, or SEQ ID NO: 67, followed by specifically identifying the fungal species. The dependent claims (within the provided summaries) further constrain amplification to PCR/real-time PCR and constrain the amplified target to internal transcribed spacer regions of nuclear ribosomal DNA, and also define fluorescent presentation formats such as a fluorochrome-dyed bead.
Stated Advantages
Provides specificity and sensitivity for fungal species identification.
Provides reduced time to diagnosis, as supported by assay performance and patient/validation results described in the document.
Documented Applications
Diagnostic methods for fungal infections using identification of specific fungal species from patient tissue or body fluid by fluorescently labeled probe hybridization to amplified fungal DNA.
Determining if a patient is at risk for or has developed a disease state related to a fungal infection based on specifically identifying the fungal species from patient tissue or body fluid.
Mycotoxin detection in patient samples using mycotoxin-specific antibodies, including ELISA/immunoaffinity and Luminex microsphere assays for mycotoxins such as aflatoxins, ochratoxin A, and tricothecenes.
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