Compositions and methods for detecting Enterovirus D68
Inventors
Nix, William Allan • Oberste, M. Steven
Assignees
US Department of Health and Human Services
Publication Number
US-9938588-B2
Publication Date
2018-04-10
Expiration Date
2036-06-03
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Abstract
Methods and compositions for detection of enterovirus D in a sample, particularly detection of enterovirus D68, are provided. The methods include contacting a sample with at least one primer (such as a forward primer and/or a reverse primer) capable of specifically amplifying an EV-D68 viral protein 1 (VP1) nucleic acid or a portion thereof and/or a detectably labeled probe capable of specifically hybridizing to an EV-D68 VP1 nucleic acid, under conditions sufficient for specific amplification of the EV-D68 VP1 nucleic acid by the at least one primer and/or under conditions sufficient for specific hybridization of the probe to the EV-D68 nucleic acid. The amplification of the EV-D68 VP1 nucleic acid and/or the hybridization of the probe to the EV-D68 VP1 nucleic acid is detected, thereby identifying presence of EV-D68 in the sample.
Core Innovation
Methods and compositions for detection of enterovirus D in a sample, particularly detection of enterovirus D68 (EV-D68), are provided. The methods include contacting a sample with at least one primer capable of specifically amplifying an EV-D68 viral protein 1 (VP1) nucleic acid or a portion thereof and/or a detectably labeled probe capable of specifically hybridizing to an EV-D68 VP1 nucleic acid, under conditions sufficient for specific amplification of the EV-D68 VP1 nucleic acid and/or specific hybridization of the probe to the EV-D68 nucleic acid. The amplification of the EV-D68 VP1 nucleic acid and/or the hybridization of the probe to the EV-D68 VP1 nucleic acid is detected, thereby identifying presence of EV-D68 in the sample.
The problem being solved is the need for specific, sensitive, and reliable detection of EV-D68, which can cause severe respiratory illness, especially in children. Since EV-D68 had been relatively rare until its re-emergence and outbreaks in the mid-2000s, including a large 2014 outbreak in the United States, there was a need for diagnostic tools that specifically detect EV-D68, particularly strains from the 2014 North America lineages, and distinguish them from other enteroviruses and related viruses.
The disclosed methods utilize primers and probes that hybridize specifically to the VP1 region of EV-D68 nucleic acids, enabling detection by amplification techniques such as real-time PCR or real-time reverse transcription-PCR (rRT-PCR). The primers and probes include sequences with at least 90% identity to SEQ ID NOs: 1-3, and the methods can specifically detect the 2014 North America EV-D68 lineage without cross-reacting with other enteroviruses or different EV-D68 lineages. Kits comprising the primers and probes are also disclosed to facilitate detection of EV-D68 in various samples.
Claims Coverage
The patent includes multiple inventive features focusing on the method of detecting EV-D68, the specific sequences of primers and probes used, and kits comprising these components.
Method for detecting EV-D68 using specific primers and probe
A method comprising contacting a sample with a forward primer (SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2) capable of amplifying an EV-D68 VP1 nucleic acid or portion thereof, and a detectably labeled probe (SEQ ID NO: 3) under conditions sufficient for amplification and hybridization, followed by detecting the amplification or hybridization, thereby determining presence of EV-D68 in the sample.
Detectably labeled probe with specific fluorophores and quenchers
Use of a detectably labeled probe comprising donor fluorophores such as FAM or CY5 and acceptor fluorophores or quenchers such as BHQ1 or BHQ2, specifically consisting of SEQ ID NO: 3 with these labeling combinations to hybridize to EV-D68 VP1 nucleic acid.
Sample types applicable for detection
The method applies to samples comprising isolated DNA, RNA, serum, nasal wash, respiratory aspirate, nasopharyngeal swab, or oropharyngeal swab for detection of EV-D68.
Isolated probe with specified sequence and label
An isolated probe consisting of the nucleic acid sequence of SEQ ID NO: 3 and a detectable label, including fluorophores, chromogenic moieties, haptens, affinity tags, or radioactive isotopes, specifically donor and acceptor fluorophores like FAM or CY5 paired with BHQ1 or BHQ2.
Kit for detecting EV-D68 comprising primers and probe
A kit containing a detectably labeled probe consisting of SEQ ID NO: 3 with a detectable label, a forward primer consisting of SEQ ID NO: 1, and a reverse primer consisting of SEQ ID NO: 2, optionally including probes and primers for human RNase P nucleic acid as internal controls.
The claims focus on a specific method of detecting human enterovirus D68 in samples using defined primer and probe sequences with particular labels, including isolated probes and kits containing these components, facilitating specific and sensitive detection of EV-D68.
Stated Advantages
High specificity for EV-D68, distinguishing it from other enteroviruses and rhinoviruses, including closely related EV-D viruses.
High sensitivity, with 100% detection of EV-D68-positive samples compared to gold-standard VP1 sequencing and detection limits comparable to established assays.
Capability to detect the 2014 North America EV-D68 lineage specifically, enabling accurate surveillance and diagnosis during outbreaks.
Use of real-time PCR and real-time RT-PCR technologies allowing for rapid and quantitative detection of EV-D68 in clinical samples.
Documented Applications
Detection of EV-D68 in biological samples from subjects suspected of infection, including respiratory samples like nasopharyngeal swabs, oropharyngeal swabs, nasal wash, respiratory aspirate, and serum.
Diagnostic and prognostic use in laboratory and clinical settings for identifying EV-D68 infection.
Use in surveillance and outbreak investigations, particularly for identifying the 2014 North America EV-D68 lineage.
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