Methods and agents for detecting Parechovirus
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsWilliam Allan Nix, M. Steven Oberste
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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.
Claims29
A method of detecting a human parechovirus (HPeV) and Ljungan virus (LV) species of Parechovirus, comprising,
- contacting cDNA reverse transcribed from RNA isolated from a sample with a composition that permits amplification of the cDNA, wherein the composition comprises a forward primer and a first reverse primer, wherein the forward and first reverse primers hybridize to opposite strands of a Parechovirus 5′ nontranslated region (5′NTR), and wherein the forward primer comprises a degenerate oligonucleotide comprising at least 95% sequence identity to the nucleic acid sequence set forth as SEQ ID NO:
- 1, and wherein the first reverse primer comprises a degenerate oligonucleotide comprising at least 95% sequence identity to the nucleic acid sequence set forth as SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- performing an amplification procedure under conditions sufficient to amplify Parechovirus cDNA and produce a Parechovirus amplicon Parechovirus, wherein the Parechovirus amplicon comprises at least a portion of the Parechovirus 5′NTR nucleic acid sequence if Parechovirus is present in the sample, thereby generating a first reaction sample;
- and
- detecting whether the Parechovirus amplicon is present in the first reaction sample, wherein the presence of the Parechovirus amplicon indicates that at least one HPeV or LV species of Parechovirus is present in the sample.
A method of identifying which Parechovirus species is present in a sample, comprising:
- contacting cDNA reverse transcribed from RNA isolated from the sample with a composition that permits amplification of the cDNA, wherein the composition comprises a first forward primer and a first reverse primer, wherein the first forward primer hybridizes to a Parechovirus VP3 region, wherein the first forward primer comprises a degenerate oligonucleotide comprising at least 95% sequence identity to the nucleic acid sequence set forth as SEQ ID NO:
- 12, and wherein the first reverse primer hybridizes to a Parechovirus 2A region, wherein the first reverse primer comprises a degenerate oligonucleotide comprising at least 95% sequence identity to the nucleic acid sequence set forth as SEQ ID NO:
- 13;
- performing an amplification procedure under conditions sufficient to amplify Parechovirus cDNA and produce a Parechovirus amplicon, wherein the Parechovirus amplicon comprises at least a portion of the Parechovirus VP1 nucleic acid sequence if Parechovirus is present in the sample, thereby generating a first reaction sample;
- amplifying at least a portion of the first reaction sample under conditions sufficient to amplify Parechovirus cDNA and produce a Parechovirus amplicon, in a composition comprising a second forward primer and a second reverse primer, wherein the second forward and reverse primers permit amplification of at least a portion of Parechovirus VP1 region, thereby generating a second reaction sample;
- and
- sequencing amplicons present in the second reaction sample, thereby generating an amplicon sequence, wherein the amplicon sequence permits a determination of which Parechovirus species is present in the sample.
A degenerate oligonucleotide consisting of any of SEQ ID NOS:
- 1-4 or 6-16.
A degenerate oligonucleotide comprising at least 95% sequence identity to SEQ ID NO:
- 2, wherein the degenerate oligonucleotide retains the ability to hybridize to a Parechovirus 5′NTR sequence under very high stringency conditions.
A degenerate oligonucleotide comprising at least 95% sequence identity to any of SEQ ID NOS:
- 6-16, wherein the degenerate oligonucleotide retains the ability to hybridize to a Parechovirus VP1 nucleic acid sequence under very high stringency conditions.
A degenerate oligonucleotide comprising at least 95% sequence identity to any of SEQ ID NOS:
- 1, 3 or 4.
Dependencies are inferred from the claim text.