Methods and agents for detecting Parechovirus
Inventors
Nix, William Allan • Oberste, M. Steven
Assignees
Centers of Disease Control and Prevention CDC • US Department of Health and Human Services
Publication Number
US-8048630-B2
Publication Date
2011-11-01
Expiration Date
2026-05-01
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The present disclosure provides methods that permit detection of all known species of Parechovirus, including Human parechovirus and Ljungan virus (LV). In particular examples the method includes amplifying at least a portion of the 5′NTR of parechovirus nucleic acid molecules obtained from a sample, and detecting the resulting amplicons, but does not require culturing of the virus. The present disclosure also provides methods for determining which particular species or serotype of parechovirus is present in a biological sample. Also provided are oligonucleotide primers and probes that can be used in the disclosed methods.
Core Innovation
The invention provides methods to detect all known species of Parechovirus, including Human parechovirus (HPeV) serotypes 1, 2, and 3, and Ljungan virus (LV), by amplifying at least a portion of the 5′ non-translated region (5′NTR) of parechovirus nucleic acid from a biological sample. The methods do not require culturing of the virus prior to detection, allowing for rapid testing with high sensitivity and specificity. The invention also discloses oligonucleotide primers and probes for use in these detection methods, as well as methods to determine the specific species or serotype of parechovirus present via sequencing of viral protein 1 (VP1).
The problem addressed is that existing diagnostic methods are unable to detect all Parechovirus species in a single assay and often rely on virus culturing, which is time-consuming, less sensitive, and costly. Prior PCR assays only detect subsets of HPeV serotypes or fail to detect LV. Furthermore, serotype identification methods require neutralization assays that rely on culturing and lack available antigenic typing reagents for LV. There is a need for rapid, sensitive, and specific detection of all known Parechovirus species directly from clinical specimens without culturing.
Claims Coverage
The patent includes 29 claims, with claims 1 and 19 as independent claims covering methods of detecting Parechovirus species and identifying the specific species present. The inventive features focus on the use of degenerate primers targeting the 5′NTR and VP1 regions of parechovirus, amplification and detection protocols, and compositions including specific primers and probes.
Method for detecting Human parechovirus and Ljungan virus species using primers targeting the 5′ nontranslated region
A method involving contacting cDNA reverse transcribed from RNA isolated from a sample with a composition containing a forward primer and a first reverse primer that hybridize to opposite strands of the Parechovirus 5′NTR, where primers comprise degenerate oligonucleotides with at least 95% identity to SEQ ID NOS: 1, 2, or 4; performing amplification to produce a Parechovirus amplicon; and detecting the amplicon to indicate presence of HPeV or LV species.
Use of a second amplification with semi-nested PCR to increase sensitivity
Performing a second amplification using the forward primer and a second reverse primer (with at least 95% identity to SEQ ID NO: 2) that hybridizes upstream of the first reverse primer on the 5′NTR to produce a second Parechovirus amplicon prior to detection.
Use of probes with fluorophore and quencher for detection
Including a probe comprising a degenerate oligonucleotide with at least 95% identity to SEQ ID NO: 3, labeled with a fluorophore and quencher to allow detection by fluorescence increase or decrease corresponding to presence or absence of Parechovirus amplicons.
Method for determining which Parechovirus species is present by VP1 gene sequencing
Amplifying at least a portion of the VP1 gene of Parechovirus using degenerate primers (at least 95% identity to SEQ ID NOS: 12-16), performing amplification including semi-nested or nested PCR, and sequencing the amplicons to compare with a sequence database containing Parechovirus VP1 sequences for species or serotype identification.
Use of degenerate oligonucleotides as primers and probes
Provision of degenerate primers and probes comprising or consisting of sequences with at least 95% sequence identity to SEQ ID NOS: 1-4 and 6-16, capable of hybridizing under very high stringency conditions to Parechovirus 5′NTR or VP1 regions.
Kits comprising multiple degenerate oligonucleotides and amplification agents
Kits including at least two degenerate oligonucleotides of the claimed sequences, optionally with agents for PCR or reverse transcription to allow performing the detection and identification methods.
The claims cover methods for sensitive and specific detection of all known Human parechoviruses and Ljungan virus species by amplifying conserved regions in the 5′NTR, employing degenerate primers and probes. They also cover subsequent identification of species or serotypes through VP1 gene amplification and sequencing, compositions of primers and probes, and kits enabling these methods without requiring virus culture.
Stated Advantages
The methods permit detection of all known Parechovirus species, including HPeV1, HPeV2, HPeV3, and Ljungan virus, in a single assay.
The methods do not require culturing of the virus, thereby reducing assay time, cost, and improving sensitivity compared to cell culture isolation.
The disclosed detection methods are approximately 10 to 100 times more sensitive than traditional virus isolation in cell culture, capable of detecting as low as one copy of the viral genome.
The methods can yield results in less than 24 hours, with specific examples ranging from about 4 hours to 13 hours depending on the assay.
The methods allow not only detection but also determination of the particular species or serotype present by viral gene sequencing.
Documented Applications
Detection of all known Parechovirus species, including Human parechovirus serotypes 1, 2, and 3, and Ljungan virus, directly from clinical specimens without culturing.
Identification of the specific Parechovirus species or serotype present in a sample by sequencing the VP1 gene region.
Diagnostic testing of various biological samples such as cerebrospinal fluid, stool, rectal swabs, nasopharyngeal swabs, tissue biopsies, and fluids from humans or non-human mammals.
Screening during suspected Parechovirus outbreaks and for clinical diagnosis of diseases associated with Parechovirus infections including meningitis, gastroenteritis, respiratory diseases, encephalitis, and neonatal sepsis-like illness.
Interested in licensing this patent?