Primers and probes for detection and discrimination of types and subtypes of influenza viruses
Inventors
Lindstrom, Stephen • Loftin, Lamorris
Assignees
Centers of Disease Control and Prevention CDC • US Department of Health and Human Services
Publication Number
US-10196699-B2
Publication Date
2019-02-05
Expiration Date
2027-02-12
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Abstract
Methods of detecting influenza, including differentiating between type and subtype are disclosed, for example to detect, type, and/or subtype an influenza infection. A sample suspected of containing a nucleic acid of an influenza virus, is screened for the presence or absence of that nucleic acid. The presence of the influenza virus nucleic acid indicates the presence of influenza virus. Determining whether the influenza virus nucleic acid is present in the sample can be accomplished by detecting hybridization between an influenza specific probe, influenza type specific probe, and/or subtype specific probe and an influenza nucleic acid. Probes and primers for the detection, typing and/or subtyping of influenza virus are also disclosed. Kits and arrays that contain the disclosed probes and/or primers also are disclosed.
Core Innovation
The invention provides methods and compositions for detecting influenza viruses, including differentiating between types and subtypes. It involves screening a sample suspected of containing influenza nucleic acid to determine the presence or absence of influenza virus nucleic acid by detecting hybridization between influenza specific probes, influenza type specific probes, and/or subtype specific probes and influenza nucleic acid. Probes and primers specific for influenza viral types and subtypes are disclosed. Kits and arrays containing these probes and primers are also included.
The background outlines the high morbidity and mortality associated with influenza infection, the importance of rapid diagnosis for effective treatment with neuraminidase inhibitors, and the limitations of current detection methods. These existing methods, such as viral culture, immunofluorescence assays, enzyme immunoassays, and serological tests, are often time-consuming, costly, or lack rapid differentiation capabilities between influenza types and subtypes, which is crucial especially for pandemic-potential strains.
The disclosed invention addresses the need for a test that provides sensitive, specific detection of influenza virus types and subtypes in a relatively short time frame to allow effective treatment. By using nucleic acid probes and primers targeting highly conserved regions of influenza viral genomes, the methods permit rapid detection, typing, and subtyping, including identification of pandemic strains such as Asian avian H5. The invention also includes arrays and kits to facilitate broad and rapid screening of samples for various influenza types and subtypes.
Claims Coverage
The claims include one independent claim directed to a specific probe and primer set, and independent method claims utilizing that set for detection and diagnosis of influenza virus nucleic acids.
Specific probe and primer set for influenza subtype H3 detection
A probe consisting of nucleotide sequences SEQ ID NO: 14, 15, or 16 with at least one detectable label, and primers consisting of the nucleic acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13, together forming a set for hybridizing specifically to influenza subtype H3 nucleic acid sequences.
Labeling of the probe with fluorophore and fluorescence quencher
The probe includes an attached label comprising a fluorophore and optionally a fluorescence quencher, with the fluorophore on the 5′-end and the fluorescence quencher on the 3′-end to enable detection of hybridization via changes in fluorescence signals.
Kit comprising the specific probe and primers for influenza detection
A kit containing the probe and primer set as described, optionally including a human RNAse P control probe and an array that comprises the probe, supporting detection of influenza nucleic acids in samples.
Method for diagnosing influenza virus infection using the probe and primer set
A method comprising contacting a sample with the described probe and primer set, amplifying influenza nucleic acid with the primers, detecting hybridization of amplified nucleic acids and the probe, and determining infection based on the detected hybridization.
Method for discriminating influenza types and subtypes
Discriminating between influenza type A and B infections and influenza subtypes H1, H3, North American H7, European H7, and Asian H9 infections by detecting hybridization of subtype-specific probes.
Detection of hybridization through change in probe signal
Detection of probe hybridization involves detecting a change in signal from the labeled probe during or after hybridization relative to before hybridization, enabling sensitive determination of target nucleic acids.
Nucleic acid amplification techniques used in detection
Amplification of influenza nucleic acids can be accomplished using PCR, real-time PCR, RT-PCR, real-time RT-PCR, ligase chain reaction, or transcription-mediated amplification (TMA).
Sample types applicable for influenza detection
Samples suitable for detection methods include bronchoalveolar lavage, tracheal aspirates, sputum, nasopharyngeal aspirates, oropharyngeal aspirates, or saliva obtained from subjects suspected of influenza infection.
Use of probe arrays for influenza detection
The probe can be arrayed in a predetermined array with addressable locations enabling simultaneous detection and identification of influenza nucleic acids in samples.
The claims collectively cover a probe and primer set specifically designed for influenza subtype H3 detection, labeled for fluorescence-based detection, incorporated into kits, and used in methods for diagnosing influenza infection by amplification and hybridization detection across diverse sample types, including discrimination among influenza types and subtypes.
Stated Advantages
Provides sensitive and specific detection of influenza virus types and subtypes rapidly to allow effective treatment.
Permits rapid differentiation between influenza types A and B and among pandemic-relevant subtypes such as H1, H3, H5, H7, and H9.
Enables detection using real-time PCR methods with high reaction efficiency, suitable for clinical and laboratory settings.
Supports use of probe arrays and kits for broad, multiplexed, and efficient influenza profiling.
Documented Applications
Diagnosing influenza infections in subjects suspected of influenza, by detecting, typing, and subtyping influenza viruses in biological samples using specific nucleic acid probes and primers.
Screening viral samples to detect and identify pandemic-potential influenza strains such as Asian avian H5, North American and European H7, and Asian H9 subtypes.
Using arrays for rapid, multiplexed identification of influenza types and subtypes in clinical and research laboratory settings.
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