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US 7,247,457 B2Granted
Detection and identification of enteroviruses by semi-nested amplification of the enterovirus VP1 protein
AssigneeThe United States of America as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention
InventorsWilliam Allan Nix, M. Steven Oberste
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Abstract
Disclosed are methods of using enterovirus-specific primers for the detection and identification of enterovirus infection. Also provided are isolated nucleic acid molecules and kits useful for detection and diagnostic testing of enterovirus infection in a subject.
Claims11
A method of detecting an enterovirus (EV) RNA sequence in a sample, comprising:
- contacting the sample with one or more nucleic acid cDNA primers comprising a sequence as set forth in any one of SEQ ID NOs:
- 1-4, that hybridize to an EV VP1 encoding sequence;
- reverse transcribing EV cDNA from the EV RNA sequence;
- amplifying at least a portion of the EV cDNA using a first nucleic acid primer pair, wherein the first forward primer comprises a sequence as set forth in SEQ ID NO:
- 5 and hybridizes to an EV VP3 encoding sequence, and the first reverse primer comprises a sequence as set forth in SEQ ID NO:
- 6 and hybridizes to an EV VP1 encoding sequence, thereby generating an EV amplicon;
- amplifying at least a portion of the EV amplicon using a second nucleic acid primer pair, wherein the second forward primer comprises a sequence as set forth in SEQ ID NO:
- 11 or SEQ ID NO:
- 13, and the second reverse primer comprises a sequence as set forth in SEQ ID NO:
- 12 or SEQ ID NO:
- 14, wherein both the second forward and reverse primers hybridize to an EV VP1 encoding sequence;
- and
- determining whether an amplified EV amplicon is present, thereby detecting an EV nucleic acid sequence in the sample.
10 more elements
A method of detecting an enterovirus (EV) RNA sequence in a sample, comprising:
- contacting the sample with one or more nucleic acid cDNA primers that hybridize to an EV VP1 encoding sequence;
- reverse transcribing EV cDNA from the EV RNA sequence;
- amplifying at least a portion of the EV cDNA using a first nucleic acid primer pair, wherein the first forward primer hybridizes to an EV VP3 encoding sequence and the first reverse primer hybridizes to an EV VP1 encoding sequence, thereby generating an EV amplicon;
- amplifying at least a portion of the EV amplicon using a second nucleic acid primer pair, wherein the second forward primer comprises a sequence as set forth in SEQ ID NO:
- 13 and the second reverse primer comprises a sequence as set forth in SEQ ID NO:12 or SEQ ID NO:14, and both second primers hybridize to an EV VP1 encoding sequence;
- and determining whether an amplified EV amplicon is present, thereby detecting an EV nucleic acid sequence in the sample.
3 more elements
A method of detecting an enterovirus (EV) RNA sequence in a sample, comprising:
- contacting the sample with one or more nucleic acid cDNA primers that hybridize to an EV VP1 encoding sequence;
- reverse transcribing EV cDNA from the EV RNA sequence;
- amplifying at least a portion of the EV cDNA using a first nucleic acid primer pair, wherein the first forward primer hybridizes to an EV VP3 encoding sequence and the first reverse primer hybridizes to an EV VP1 encoding sequence, thereby generating an EV amplicon;
- amplifying at least a portion of the EV amplicon using a second nucleic acid primer pair, wherein the second forward primer comprises a sequence as set forth in SEQ ID NO:11 or SEQ ID NO:13 and the second reverse primer comprises a sequence as set forth in SEQ ID NO:14, and both second primers hybridize to an EV VP1 encoding sequence;
- and determining whether an amplified EV amplicon is present, thereby detecting an EV nucleic acid sequence in the sample.
2 more elements
Dependencies are inferred from the claim text.