Sensitive RT-qPCR for Zika Virus Detection

ZIKV RNA was isolated from both fluid and tissue samples as previously described [17 (link)]. Viral RNA was then quantified using a highly sensitive RT-qPCR assay based on the one developed by Lanciotti et al. [25 (link)], though the primers were modified with degenerate bases at select sites to accommodate African-lineage Zika viruses. RNA was reverse-transcribed and amplified using the TaqMan Fast Virus 1-Step Master Mix RT-qPCR kit (Life Technologies, Carlsbad, CA) on the LightCycler 480 or LC96 instrument (Roche, Indianapolis, IN), and quantified by interpolation onto a standard curve made up of serial tenfold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from a plasmid containing an 800 bp region of the Zika virus genome that is targeted by the RT-qPCR assay. The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5’- CGYTGCCCAACACAAGG-3’, reverse primer: 5′-CCACYAAYGTTCTTTTGCABACAT-3′ and probe: 5′-6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA. The limit of detection for fluids (plasma, amniotic fluid, CSF) with this assay is 150 copies/ml. The theoretical limit of detection for the tissues is 3 copies/mg [18 (link)].