title: Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses.
abstract: Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies.
authors.affiliation: Diagnostic Systems Division, and Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21701-5011, USA.
authors.forename: Adrienne R
authors.initials: AR
authors.lastname: Trombley
authors.forename: Leslie
authors.initials: L
authors.lastname: Wachter
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Garrison
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Buckley-Beason
authors.forename: Jordan
authors.initials: J
authors.lastname: Jahrling
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Hensley
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Schoepp
authors.forename: David A
authors.initials: DA
authors.lastname: Norwood
authors.forename: Augustine
authors.initials: A
authors.lastname: Goba
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Fair
authors.forename: David A
authors.initials: DA
authors.lastname: Kulesh
mesh_terms: D018050:Arenavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016563:Filoviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006476:Orthohantavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000145:classification / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral