title: Evaluation of a 384-well format for high-throughput real-time reverse transcription polymerase chain reaction for avian influenza testing.
abstract: As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384-well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96- and 384-well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96- and 384-well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96- and 384-well assays, respectively. The data presented herein supports the utility of the 384-well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Ferro
authors.forename: Jason
authors.initials: J
authors.lastname: Osterstock
authors.forename: Bo
authors.initials: B
authors.lastname: Norby
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Fosgate
authors.forename: Blanca
authors.initials: B
authors.lastname: Lupiani
mesh_terms: D000818:Animals; D001717:Birds; D002140:California / Q000453:epidemiology; D002988:Cloaca / Q000821:virology; D004196:Disease Outbreaks / Q000662:veterinary; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification; D005585:Influenza in Birds / Q000453:epidemiology / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary*; D012680:Sensitivity and Specificity