title: A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.
abstract: CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).
authors.affiliation: Department of Medicine, Division of Infectious Diseases, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Sanchita
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Rundell
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Aashiq H
authors.initials: AH
authors.lastname: Mirza
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Maneesh R
authors.initials: MR
authors.lastname: Pingle
authors.affiliation: Department of Medicine, Division of Infectious Diseases, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Kristi
authors.initials: K
authors.lastname: Shigyo
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, United States of America.
authors.forename: Aura R
authors.initials: AR
authors.lastname: Garrison
authors.affiliation: Integrated Research Facility, Division of Clinical Research, NIAID, NIH, Fort Detrick, Maryland, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Paragas
authors.affiliation: Poxvirus Team, Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, National Center of Emerging Zoonotic and Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Scott K
authors.initials: SK
authors.lastname: Smith
authors.affiliation: Poxvirus Team, Poxvirus and Rabies Branch, Division of High Consequence Pathogens and Pathology, National Center of Emerging Zoonotic and Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
authors.forename: Victoria A
authors.initials: VA
authors.lastname: Olson
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America; Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, New York, NY, United States of America.
authors.forename: Davise H
authors.initials: DH
authors.lastname: Larone
authors.affiliation: Department of Pathology, Stony Brook University Medical Center, Stony Brook, New York, United States of America.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Spitzer
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Francis
authors.initials: F
authors.lastname: Barany
authors.affiliation: Department of Medicine, Division of Infectious Diseases, Weill Medical College of Cornell University, New York, New York, United States of America; Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America.
authors.forename: Linnie M
authors.initials: LM
authors.lastname: Golightly
mesh_terms: D006482:Hemorrhagic Fevers, Viral / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012899:Smallpox / Q000175:diagnosis* / Q000821:virology; D012901:Variola virus / Q000302:isolation & purification*; D014780:Viruses / Q000302:isolation & purification*