Compositions and methods for detection of neisseria gonorroheae
Inventors
FISS HOBART, Ellen H. • Sun, Jingtao
Assignees
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Publication Number
US-12385101-B2
Publication Date
2025-08-12
Expiration Date
Abstract
Methods for the rapid detection of the presence or absence of NG Pilin Inverting protein (PivNg) gene 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 NG PivNg genes, along with kits are provided that are designed for the detection of Neisseria gonorrhoeae (NG).
Core Innovation
The invention provides a method of detecting Neisseria gonorrhoeae in a sample by performing an amplifying step and a hybridizing/detecting workflow that distinguishes presence from absence. The amplifying step contacts the sample with a set of NG PivNg gene primers to produce an amplification product if NG PivNg nucleic acid is present, and the subsequent hybridizing step contacts the amplification product with one or more detectable NG PivNg gene probes.
The disclosed approach grounds specificity in particular NG PivNg gene primer and probe sequences, where the set of NG PivNg gene primers includes a first primer with an oligonucleotide sequence of SEQ ID No: 1 or a complement and a second primer with an oligonucleotide sequence of SEQ ID No: 2 or a complement. The one or more detectable NG PivNg gene probes includes a third oligonucleotide sequence of SEQ ID No: 3 or a complement, linking amplification-product detection directly to the presence or absence of NG PivNg nucleic acid.
Dependent embodiments describe fluorescence resonance energy transfer detection by detecting fluorescence associated with donor and acceptor fluorescent moieties on the probe, including embodiments where an acceptor quencher is present and where donor and acceptor moieties are positioned within a defined nucleotide spacing on the probe. Other embodiments specify a polymerase with 5′ to 3′ nuclease activity, modified nucleotides selected from N6-benzyl-dA, N4-benzyl-dC, N6-para-tert-butyl-benzyl-dA, or N4-para-tert-butyl-benzyl-dC, and multiplex detection with primers and probes for the NGDR9 gene sequence.
Claims Coverage
The independent claim covers NG detection by amplification of PivNg nucleic acid using specified SEQ ID No: 1 and SEQ ID No: 2 primers, hybridization with a detectable probe having SEQ ID No: 3, and detection of whether an amplification product is present to indicate NG presence or absence. The dependent claims add FRET readout, donor/acceptor proximity constraints, polymerase 5′ to 3′ nuclease activity, specified modified nucleotide options, and optional multiplexing with the NGDR9 gene sequence.
PivNg primer set and amplification-product presence/absence detection
Contacting the sample with a set of NG PivNg gene primers comprising a first primer with an oligonucleotide sequence of SEQ ID No: 1 or a complement thereof and a second primer with an oligonucleotide sequence of SEQ ID No: 2 or a complement thereof to produce an amplification product if NG PivNg nucleic acid is present, and detecting the presence or absence of the amplification product to indicate NG presence or absence.
Detectable PivNg probe hybridization for NG indication
Contacting the amplification product with one or more detectable NG PivNg gene probes comprising a third oligonucleotide sequence of SEQ ID No: 3 or the complement thereof, and detecting whether the amplification product is present to indicate the presence or absence of NG in the sample.
FRET-based detection using donor and acceptor moieties on the probe
Hybridizing the amplification product with a labeled NG PivNg gene probe and detecting fluorescence resonance energy transfer fluorescence between a donor fluorescent moiety and an acceptor moiety to indicate whether NG is present in the sample.
Donor-acceptor proximity constraint on the probe
Positioning a donor fluorescent moiety and a corresponding acceptor moiety within 8 to 20 nucleotides of each other on the probe.
Polymerase enzyme with 5′ to 3′ nuclease activity in amplification
Specifying that the amplifying step uses a polymerase enzyme with 5′ to 3′ nuclease activity.
Modified nucleotides selected from specific substituted adenine/cytosine forms
Defining that at least one modified nucleotide is selected from N6-benzyl-dA, N4-benzyl-dC, N6-para-tert-butyl-benzyl-dA, or N4-para-tert-butyl-benzyl-dC.
Optional multiplex detection of the NGDR9 gene sequence
Contacting a sample with a set of primers and probes to amplify and detect the NGDR9 gene sequence in addition to the NG PivNg gene detection.
Across the independent and dependent claims, the coverage centers on detecting NG by producing an amplification product from NG PivNg nucleic acid using the specified SEQ ID No: 1 and SEQ ID No: 2 primers, hybridizing with the SEQ ID No: 3 detectable probe, and calling NG based on the presence or absence of the amplification product. Dependent claims further refine the assay by using FRET readout, donor/acceptor spacing, polymerase 5′ to 3′ nuclease activity, specified modified nucleotide options, and optional multiplexing with the NGDR9 gene sequence.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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