title: Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols.
abstract: Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.
authors.affiliation: Diagnostic Virology Laboratory, National Veterinary Services Laboratories, VS, APHIS, USDA, Ames, Iowa 50010, USA.
authors.forename: Janet V
authors.initials: JV
authors.lastname: Warg
authors.forename: Travis
authors.initials: T
authors.lastname: Clement
authors.forename: Emily R
authors.initials: ER
authors.lastname: Cornwell
authors.forename: Angela
authors.initials: A
authors.lastname: Cruz
authors.forename: Rodman G
authors.initials: RG
authors.lastname: Getchell
authors.forename: Cem
authors.initials: C
authors.lastname: Giray
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Goodwin
authors.forename: Geoffrey H
authors.initials: GH
authors.lastname: Groocock
authors.forename: Mohamed
authors.initials: M
authors.lastname: Faisal
authors.forename: Robert
authors.initials: R
authors.lastname: Kim
authors.forename: Gwenn E
authors.initials: GE
authors.lastname: Merry
authors.forename: Nicholas B D
authors.initials: NB
authors.lastname: Phelps
authors.forename: Monica M
authors.initials: MM
authors.lastname: Reising
authors.forename: Isaac
authors.initials: I
authors.lastname: Standish
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Kathy
authors.initials: K
authors.lastname: Toohey-Kurth
mesh_terms: D000818:Animals; D005399:Fishes; D005838:Genotype; D031941:Hemorrhagic Septicemia, Viral / Q000821:virology*; D029141:Novirhabdovirus / Q000235:genetics / Q000302:isolation & purification*; D011159:Population Surveillance; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity