Abstract |
Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses ( variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.
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Authors | S L Ropp, Q Jin, J C Knight, R F Massung, J J Esposito |
Journal | Journal of clinical microbiology
(J Clin Microbiol)
Vol. 33
Issue 8
Pg. 2069-76
(Aug 1995)
ISSN: 0095-1137 [Print] United States |
PMID | 7559950
(Publication Type: Comparative Study, Journal Article)
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Chemical References |
- DNA Primers
- DNA, Viral
- Hemagglutinins, Viral
- DNA Restriction Enzymes
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Topics |
- Animals
- Base Sequence
- Consensus Sequence
- DNA Primers
(genetics)
- DNA Restriction Enzymes
- DNA, Viral
(genetics)
- Evaluation Studies as Topic
- Hemagglutinins, Viral
(genetics)
- Molecular Sequence Data
- Open Reading Frames
- Orthopoxvirus
(classification, genetics)
- Polymerase Chain Reaction
(methods)
- Species Specificity
- Variola virus
(classification, genetics)
- Virology
(methods)
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