Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing
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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.
author list (cited authors)
Ferro, P. J., Osterstock, J., Norby, B. o., Fosgate, G. T., & Lupiani, B.