title: Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans.
abstract: Smallpox, once a devastating disease caused by Variola virus, a member of the Orthopoxvirus genus, was eradicated in 1980. However, the importance of variola virus infections has been stressed widely in the last few years, particularly following recent social events in the world. Today, variola virus is considered to be one of the most significant agents with potential use as a biological weapon. In this study we developed an internally controlled real-time PCR assay for rapid detection and simultaneous differentiation of variola virus from other orthopoxviruses. The assay is based on TaqMan 3'-minor groove binder (MGB) chemistry and uses generic primers, designed in highly conserved genomic regions of the crmB gene, and three TaqMan MGB probes designed to identify orthopoxviruses, variola virus, and an internal control. The results obtained suggest that the assay is rapid, sensitive, specific, and suitable for the generic detection of orthopoxviruses and the identification of variola virus and avoids false-negative results in a single reaction tube.
authors.affiliation: Alerts and Emergencies Unit, National Center of Microbiology, Instituto de Salud Carlos III, Ctra. Pozuelo Majadahonda Km 2, 28220 Majadahonda, Madrid, Spain. gfedele@isciii.es
authors.forename: C G
authors.initials: CG
authors.lastname: Fedele
authors.forename: A
authors.initials: A
authors.lastname: Negredo
authors.forename: F
authors.initials: F
authors.lastname: Molero
authors.forename: M P
authors.initials: MP
authors.lastname: Sánchez-Seco
authors.forename: A
authors.initials: A
authors.lastname: Tenorio
mesh_terms: D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D016679:Genome, Viral; D006801:Humans; D018155:Orthopoxvirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D011786:Quality Control; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012901:Variola virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D018124:Receptors, Tumor Necrosis Factor; D014764:Viral Proteins; C089458:crmB protein, Orthopoxvirus