Rapid and high-throughput pan-Orthopoxvirus detection and identification using PCR and mass spectrometry.
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.
Eshoo
Mark W
MW
Ibis Biosciences, Carlsbad, CA, USA. meshoo@ibisbio.com
Whitehouse
Chris A
CA
Nalca
Aysegul
A
Zoll
Scott
S
Ecker
Joseph A
JA
Hall
Thomas A
TA
Pennella
Thuy-Trang D
TT
Duncan
David D
DD
Desai
Anjali
A
Moradi
Emily K
EK
Rudnick
Karl
K
Libby
Brian
B
Ranken
Raymond
R
Sampath
Rangarajan
R
Hofstadler
Steven A
SA
Ecker
David J
DJ
Blyn
Lawrence B
LB
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
D017931:DNA Primers; D004279:DNA, Viral