Lc96 instrument

ZIKV RNA was isolated from both fluid and tissue samples as previously described [39 (link)]. Viral RNA was then quantified using a highly sensitive RT-qPCR assay based on the one developed by Lanciotti et al. [57 (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, USA) on the LC96 instrument (Roche, Indianapolis, IN, USA), and quantified by interpolation onto a standard curve made up of serial 10-fold 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, extraembryonic coelomic fluid, amniotic fluid, CSF) with this assay is 150 copies/ml. The theoretical limit of detection for the tissues is 3 copies/mg [23 (link)].

Quantitative PCR for P. falciparum 18S rDNA and human actin was performed as a multiplexed reaction using Qiagen QuantiTect multiplex RTPCR master mix and a Roche LC96 instrument. In the case of reverse-transcriptase PCR, Qiagen RT-MIX was added to the master mix and a RT step consisting of 50 °C for 20 min was included prior to PCR. All cycling conditions and primer/probe sequences were identical as previously reported [14 (link)]. Experiments were typically performed with at least three biological replicates and two technical replicates. An identical k13 PCR protocol was followed as previously reported [8 (link)]. PCR products were run on 2% agarose gels and stained with ethidium bromide.