Compositions and methods for detection of Candida auris
Inventors
FISS HOBART, Ellen H. • Harris, Jody • Hill, Andrew T. • Sun, Jingtao
Assignees
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Publication Number
US-12344905-B2
Publication Date
2025-07-01
Expiration Date
Abstract
Methods for the rapid detection of the presence or absence of Candida auris (CA) in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers, probes targeting the CA 5.8s/ITS2 rRNA gene, along with kits are provided that are designed for the detection of CA.
Core Innovation
The invention relates to molecular diagnostics for detecting Candida auris (CA) in a sample while discriminating against Candida haemulonii (CH). The method uses CA 5.8s/ITS2 rRNA gene nucleic acid as the target and determines CA presence or absence based on detecting the presence or absence of a CA amplification product.
The approach performs an amplifying step using a set of CA 5.8s/ITS2 rRNA gene primers to produce an amplification product if CA 5.8s/ITS2 rRNA nucleic acid is present. A hybridizing step then contacts the amplification product with one or more detectable CA 5.8s/ITS2 rRNA gene probes, followed by detecting the presence or absence of the amplification product to indicate CA presence or absence.
A key feature is that the combination of the CA 5.8s/ITS2 rRNA gene primers and the detectably labeled probe shows no detectable cross-reactivity with Candida haemulonii in a PCR amplification reaction. In described embodiments, fluorescence detection includes a donor/acceptor arrangement configured for FRET/hydrolysis probe formats, and optional melting-curve confirmation is described for double-stranded DNA dye detection.
Claims Coverage
Two independent claims are directed to a CA detection method that discriminates against CH using CA 5.8s/ITS2 rRNA gene primer/probe sets with no detectable CH cross-reactivity, and a kit comprising the corresponding primer and fluorescently detectably labeled probes configured to detect CA nucleic acid with the same no detectable CH cross-reactivity limitation.
CA 5.8s/ITS2 rRNA gene primer set for discriminating CA from CH
The set of CA 5.8s/ITS2 rRNA gene primers comprises a first oligonucleotide primer comprising a first nucleic acid sequence selected from SEQ ID NOs: 1-3, and a second oligonucleotide primer comprising a second nucleic acid sequence selected from SEQ ID NOs: 5-6, and 8; the combination exhibit no detectable cross-reactivity with Candida haemulonii in a PCR amplification reaction.
CA 5.8s/ITS2 rRNA gene probe for hybridizing detection
The one or more detectable CA 5.8s/ITS2 rRNA gene oligonucleotide probe comprises a third nucleic acid sequence selected from SEQ ID NOs: 9-11, or the complement thereof, configured to hybridize to an amplification product generated by the first and the second oligonucleotide primer; detecting presence of the amplification product indicates CA presence and detecting absence indicates CA absence.
FRET-based or fluorescently detectably labeled probe detection format
The detectably labeled probe is configured for fluorescence detection, including donor fluorescent moiety and corresponding acceptor moiety, and is described as exhibiting donor/acceptor arrangements for FRET-based fluorescence indicating CA presence/absence.
Kit for detecting CA nucleic acid with no detectable CH cross-reactivity
A kit comprising: a first oligonucleotide primer comprising a first nucleic acid sequence selected from SEQ ID NOs: 1-3; a second oligonucleotide primer comprising a second nucleic acid sequence selected from SEQ ID NOs: 5-6, and 8; and a fluorescently detectably labeled oligonucleotide probe comprising a third nucleic acid sequence selected from SEQ ID NOs: 9-11, or a complement thereof, configured to hybridize to an amplicon generated by the first and the second oligonucleotide primer, wherein the combination exhibit no detectable cross-reactivity with Candida haemulonii in a PCR amplification reaction.
The claimed scope centers on CA 5.8s/ITS2 rRNA gene amplification followed by hybridization with CA 5.8s/ITS2 rRNA gene probes, where specific primer/probe sequence combinations from defined SEQ ID ranges are required to show no detectable cross-reactivity with Candida haemulonii. The kit claim mirrors the same primer/probe combination in a packaged format, with fluorescence-based probe detection and described donor/acceptor, including FRET, probe labeling features.
Stated Advantages
Discriminates CA from Candida haemulonii by exhibiting no detectable cross-reactivity with Candida haemulonii in a PCR amplification reaction.
Provides detection of CA presence or absence based on whether an amplification product is detected.
Documented Applications
Rapid molecular detection of Candida auris (CA) in a sample by real-time PCR using CA 5.8s/ITS2 rRNA gene primers and detectable CA 5.8s/ITS2 rRNA gene probes.
Assay use to determine CA presence/absence by detecting fluorescence signals associated with probe hybridization/amplification products, including described FRET/hydrolysis-probe fluorescence formats and optional double-stranded DNA dye melting-curve confirmation.
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