Methods and compositions for detecting Aspergillus terreus, Aspergillus niger, and mycotoxins
Inventors
Assignees
Mycodart Inc • MEDICAL SERVICE CONSULTATION INTERNATIONAL LLC
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Publication Number
US-9150934-B2
Publication Date
2015-10-06
Expiration Date
Abstract
The invention relates to a method of identifying an Aspergillus terreus or an Aspergillus niger fungal species in patient tissue or body fluid and to primers and probes for use in such a method.
Core Innovation
The invention relates to diagnostic methods for identifying Aspergillus niger fungal species in a patient tissue or a patient body fluid. The method extracts and recovers DNA from the patient tissue or the patient body fluid, amplifies the recovered DNA, and hybridizes a probe to the amplified DNA for specifically identifying the fungal species.
The probe consists of SEQ ID NO: 4 for Aspergillus niger, and the amplified DNA is specifically identified based on probe hybridization. The disclosed approach uses fluorescently labeled probes and bead-based fluorochrome formats, with target specificity supported by internal transcribed spacer regions of nuclear ribosomal DNA.
Specificity testing results select Design 1 for Aspergillus niger, and the selected design detects only Aspergillus niger among the compared Aspergillus species, including A. flavus and A. fumigatus, using the disclosed primer and probe set. The document also discloses kits and purified nucleic acids comprising the disclosed SEQ ID NOs for use in the diagnostic approach.
Claims Coverage
The independent claims cover an Aspergillus niger identification method in patient tissue or patient body fluid using DNA extraction, DNA amplification, and probe hybridization with a probe consisting of SEQ ID NO: 4, followed by specific fungal species identification. Across the dependent refinements referenced, the inventive features add species-specific primers, optional real-time PCR, fluorescent probe and bead formats, internal transcribed spacer regions of nuclear ribosomal DNA, and restriction of allowable body fluid types.
Aspergillus niger species identification by SEQ ID NO: 4 probe hybridization
A method of identifying an Aspergillus niger fungal species in a patient tissue or a patient body fluid by extracting and recovering Aspergillus niger DNA, amplifying the DNA, hybridizing a probe consisting of SEQ ID NO: 4 to the DNA, and specifically identifying the Aspergillus niger fungal species.
Species-specific amplification primers for Aspergillus niger detection
The method further amplifies DNA using a forward primer consisting of SEQ ID NO: 5 and a reverse primer consisting of SEQ ID NO: 6.
Real-time PCR amplification
The method is performed using real-time PCR.
Fluorescently labeled probe
The method is performed such that the probe is fluorescently labeled.
Targeting internal transcribed spacer regions of nuclear ribosomal DNA
The method is performed such that the amplified DNA comprises internal transcribed spacer regions of nuclear ribosomal DNA.
Restricted sample types from specified patient body fluids
The method further defines selecting the body fluid from a specified group of bodily fluids.
Overall, the claim coverage centers on hybridization-based identification of Aspergillus niger using a probe consisting of SEQ ID NO: 4, with dependent features specifying SEQ ID NO: 5 and SEQ ID NO: 6 primers, optional real-time PCR, fluorescent labeling and bead-based fluorochrome formats, internal transcribed spacer regions of nuclear ribosomal DNA as the amplified target, and restriction of allowable patient body fluid types.
Stated Advantages
The selected design detects only Aspergillus niger among the compared Aspergillus species, including A. flavus and A. fumigatus.
Documented Applications
Diagnostic methods for identifying Aspergillus niger in patient tissue or patient body fluid.
Diagnostic monitoring.
Kits and purified nucleic acids comprising the disclosed SEQ ID NOs for use in the diagnostic approach.
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