Method Description

For this real-time reverse-transcription laboratory-developed polymerase chain reaction (PCR) assay, viral nucleic acid is extracted from specimens, followed by amplification and detection on the Roche LightCycler 480 instrument. This PCR assay has been optimized to detect a target sequence in the nonstructural protein. Primers amplify a 179 base pair product. Powassan virus genomic RNA is first transcribed to complementary DNA (cDNA) by reverse transcriptase, followed by amplification of the cDNA product. The LightCycler 480 is an automated instrument that amplifies and monitors the development of target nucleic acid (amplicon) after each cycle of PCR. The detection of amplicon is based on fluorescence resonance energy transfer, which utilizes a hybridization probe with a donor fluorophore, fluorescein, at the 3' end and a second hybridization probe with an acceptor fluorophore, LC-Red 610, at the 5' end. When the target amplicon is present, the LC-Red 610 emits a measurable and quantifiable light signal at a specific wavelength. Presence of the specific organism nucleic acid may be confirmed by performing a melting curve analysis of the amplicon. Using features of the melting curve analysis, the assay primers and specific hybridization probes are able to detect Powassan virus lineage 1 and lineage 2.(Unpublished Mayo method)