title: Rapid and high-throughput pan-Orthopoxvirus detection and identification using PCR and mass spectrometry.
abstract: The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit.
authors.affiliation: Ibis Biosciences, Carlsbad, CA, USA. meshoo@ibisbio.com
authors.forename: Mark W
authors.initials: MW
authors.lastname: Eshoo
authors.forename: Chris A
authors.initials: CA
authors.lastname: Whitehouse
authors.forename: Aysegul
authors.initials: A
authors.lastname: Nalca
authors.forename: Scott
authors.initials: S
authors.lastname: Zoll
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Ecker
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Hall
authors.forename: Thuy-Trang D
authors.initials: TT
authors.lastname: Pennella
authors.forename: David D
authors.initials: DD
authors.lastname: Duncan
authors.forename: Anjali
authors.initials: A
authors.lastname: Desai
authors.forename: Emily K
authors.initials: EK
authors.lastname: Moradi
authors.forename: Karl
authors.initials: K
authors.lastname: Rudnick
authors.forename: Brian
authors.initials: B
authors.lastname: Libby
authors.forename: Raymond
authors.initials: R
authors.lastname: Ranken
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sampath
authors.forename: Steven A
authors.initials: SA
authors.lastname: Hofstadler
authors.forename: David J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Blyn
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D018155:Orthopoxvirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D004279:DNA, Viral