title: Detection of vaccinia virus DNA on the LightCycler by fluorescence melting curve analysis.
abstract: After eradication of variola virus the worldwide vaccination program was stopped to avoid the severe complications observed in a small fraction of vaccinees. Hence, there is at least one non-vaccinated generation in the human population that is immunologically naive with respect to variola virus infections. The possibility of a deliberate release of variola virus by bioterrorist attacks has led to the resumption of vaccination of hospital employees and military personnel with vaccinia virus in certain parts of the world. However, the appearance of a single confirmed smallpox case worldwide would result in vaccination of possible contact persons with vaccinia virus. Therefore, reliable confirmation of vaccinia virus in patients presenting with smallpox-like syndromes is required. A vaccinia virus-specific single nucleotide polymorphism was identified in the gene B8R coding for a vaccinia virus IFNgamma receptor. Based on this polymorphism, the LightCycler real-time PCR assay detects vaccinia virus DNA in a linear range from 10(6) to 10 genome equivalents and discriminates vaccinia virus from other orthopoxviruses by fluorescence melting curve analysis (DeltaT = 9 degrees C). While the assay amplifies generically DNA of all orthopoxviruses tested, amplification curves are only displayed for vaccinia virus strains including strains formerly used for vaccination. In addition, an internal amplification control is described that allows reliable interpretation of results.
authors.affiliation: Robert Koch-Institut, Zentrum für Biologische Sicherheit 1, Nordufer 20, D-13353 Berlin, Germany. nitschea@rki.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Nitsche
authors.forename: Brigitte
authors.initials: B
authors.lastname: Steger
authors.forename: Heinz
authors.initials: H
authors.lastname: Ellerbrok
authors.forename: Georg
authors.initials: G
authors.lastname: Pauli
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D012015:Reference Standards; D044366:Transition Temperature; D014616:Vaccinia virus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004279:DNA, Viral