Viral RNA was quantified using a highly sensitive RT-qPCR assay based on the one developed by Lanciotti et al.[49 (link)], though the primers were modified to accommodate both Asian and African lineage ZIKV lineages. Each sample was run in duplicate. RNA was reverse transcribed and amplified using the TaqMan Fast Virus 1-Step Master Mix RT-qPCR kit (Invitrogen) on a 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 base pair region of the ZIKV genome 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-BHQ1-3’.
The reactions cycled with the following conditions: 50°C for 5 minutes, 95°C for 20 seconds followed by 50 cycles of 95°C for 15 seconds, and 60°C for 1 min. The limit of detection of this assay in body fluids is 150 copies/ml and 3 copies/mg in tissues.
forward primer: 5’-CGYTGCCCAACACAAGG-3’
reverse primer: 5′-CCACYAAYGTTCTTTTGCABACAT-3′
and probe: 5′-6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA-BHQ1-3’.
The reactions cycled with the following conditions: 50°C for 5 minutes, 95°C for 20 seconds followed by 50 cycles of 95°C for 15 seconds, and 60°C for 1 min. The limit of detection of this assay in body fluids is 150 copies/ml and 3 copies/mg in tissues.
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