Compositions and methods for detection and discrimination of emerging influenza virus subtypes
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10329630-B2
Publication Date
2019-06-25
Expiration Date
2037-12-07
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Abstract
Compositions and methods for detecting presence of an emerging influenza virus in a sample, such as a biological sample obtained from a subject or an environmental sample, are disclosed. In some embodiments, the compositions and methods can be used to quickly identify particular subtypes of influenza virus (such as a pandemic and/or emerging influenza virus subtype). Probes and primers are provided herein that permit the rapid detection and/or discrimination of pandemic influenza virus subtype nucleic acids in a sample. Devices (such as arrays) and kits for detection and/or discrimination of influenza virus subtype nucleic acids are also provided.
Core Innovation
The invention relates to compositions and methods for detecting the presence of an influenza virus in a sample, such as biological or environmental samples. It provides probes and primers that enable the rapid detection and discrimination of influenza virus subtypes, including pandemic and emerging subtypes like the 2009 pandemic influenza A subtype H1. Devices such as arrays and kits containing these probes and primers are also disclosed for identification of influenza nucleic acids in samples.
The problem being solved addresses the need for sensitive and specific tests that can detect and subtype influenza viruses rapidly to allow timely treatment and management of infections. Influenza A viruses have a substantial pandemic potential due to genetic reassortment, resulting in novel subtypes to which humans lack immunity. Existing assays were shown to fail in detecting certain emerging influenza virus strains with nucleotide substitutions, underlining the need for updated and optimized assays with increased sensitivity and specificity.
Claims Coverage
There are seven main inventive features disclosed in seven independent claims, covering kit compositions and methods for detecting influenza virus nucleic acids using specific probes and primers.
Kit comprising defined probe and primers for influenza virus detection
A kit includes a probe consisting of SEQ ID NO: 3 with at least one detectable label (radioactive isotope, enzyme substrate, co-factor, ligand, chemiluminescent agent, fluorophore, fluorescence quencher, hapten, enzyme, chemical, or combinations thereof), a forward primer consisting of SEQ ID NO: 1, and a reverse primer consisting of SEQ ID NO: 2.
Kit further comprising additional influenza type A probes and primers
The kit further includes one or both of: (a) a detectably labeled probe of SEQ ID NO: 6 with a forward primer SEQ ID NO: 4 and reverse primer SEQ ID NO: 5; and/or (b) a detectably labeled probe of SEQ ID NO: 9 with a forward primer SEQ ID NO: 7 and reverse primer SEQ ID NO: 8.
Kit including probes and primers for multiple influenza subtypes
The kit further contains one or more of probes and primer pairs for various influenza subtypes, including SEQ ID NOs for probes (12, 13, 18, 19, 22, 25, 28, 31, 34) and primers (10/11, 14/15, 16/17, 20/21, 23/24, 26/27, 29/30, 32/33), or combinations thereof.
Kit including a human RNAse P control probe
The kit further includes a human RNAse P control probe for assay control.
Kit expanding claim 2 with probes and primers consisting exactly of defined sequences
The kit includes detectably labeled probes and primers exactly consisting of SEQ ID NO: 6, 4, 5 and/or SEQ ID NO: 9, 7, 8.
Kit expanding claim 3 with probes and primers consisting exactly of defined sequences
The kit includes probes and primer pairs exactly consisting of SEQ ID NOs: 10-34 for various influenza subtypes.
Method for detecting influenza virus nucleic acid using the kit components
A method comprises contacting nucleic acid from a sample with the kit's probe and primers, amplifying the nucleic acid by PCR or variants thereof, and detecting hybridization between the labeled probe and influenza virus nucleic acid. Detection indicates the virus's presence in the sample.
The claims cover kits comprising specific influenza subtype H1 pandemic 2009 nucleic acid probes and primers, additional influenza type and subtype probes and primers, control probes, and methods of using these kits with amplification and hybridization detection techniques to identify influenza virus nucleic acids in biological or environmental samples.
Stated Advantages
The updated probe and primer sets provide sensitive and specific detection of pandemic and emerging influenza virus subtypes, including strains with nucleotide mutations undetectable by previous assays.
The methods enable rapid detection and discrimination of multiple influenza subtypes, facilitating timely diagnosis and effective treatment.
The use of labeled probes and real-time PCR allows for quantification and real-time monitoring of amplification, improving detection accuracy and efficiency.
The assay redesign improves inclusivity by detecting influenza variants that might evade prior detection, reducing the risk of missing circulating and emerging influenza viruses.
Kits and arrays enable multiplexed and diagnostic testing with internal controls, improving reliability and adaptability to clinical and environmental surveillance settings.
Documented Applications
Diagnosing influenza infections in subjects suspected of having influenza by analyzing biological specimens.
Detection and subtyping of influenza viruses in environmental samples to monitor contamination.
Rapid identification and discrimination of pandemic and emerging influenza virus subtypes, including 2009 pandemic influenza A subtype H1, seasonal and variant subtypes H3, H5, Eurasian and North American H7, and H9 subtypes.
Use of kits and arrays for surveillance of influenza in humans, avian and swine populations, facilitating detection of novel reassortant viruses.
Multiplex detection of additional respiratory viral and bacterial nucleic acids in conjunction with influenza detection, including rhinovirus, coronavirus, respiratory syncytial virus, adenovirus, parainfluenza, and respiratory bacteria such as Legionella and Haemophilus influenzae.
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