Test: Powassan Virus (POWV) PCR Acute
The reverse transcription polymerase chain reaction (RT-PCR) is one of the many variants of PCR used in molecular diagnostics. This technique is commonly used to detect viruses with an RNA genome. RT-PCR is used to clone expressed genes by reverse transcribing the RNA sequence of interest into its DNA complement (cDNA) through the use of the enzyme reverse transcriptase. Subsequently, the newly synthesized cDNA can then be amplified using traditional PCR methods.
This procedure detects the presence of Powassan virus lineage I (POWV) and lineage II (DTV) genomic RNA in human blood samples. Total nucleic acid from a human sample is purified. A viral gene of interest is reverse transcribed and the cDNA created is then amplified using primers specific for the virus. The presence of specific POWV / DTV amplicon allows a TaqMan probe to bind and be cleaved during amplification. As amplicon quantity increases, the fluorescent signal increases. If the sample is positive the fluorescent signal will pass a pre-determined threshold. The cycle at which the fluorescence passes the threshold is compared to a standard curve of synthetic double-stranded DNA (DSD) to determine a relative quantity of POWV / DTV RNA in the sample. If no amplicon is created, then no probe will be cleaved providing no fluorescent signal. Calibrator, medium positive, low positive and negative control reactions are performed with each test.