ZIKV Genomic RNA Detection by RT-qPCR

ZIKV genomic RNA was detected using the GoTaq^® Probe 1-Step RT-qPCR System (Promega, Dalian, China), including forward primer: 5′-AAGTTTGCATGCTCCAAGAAAAT-3′, reverse primer: 5′-CAGCATTATCCGGTACTCCAGAT-3′ and probe: 5′-FAM-ACCGGGAAGAGCATCCAGCCAGA-TAMRA-3′ [36 (link)]. The following reagents were used for the RT-qPCR reactions: 2 μL of RNA sample, 10 μL of GoTaq^® Probe qPCR Master Mix, 0.4 μL of GoScript™ RT Mix, 1 μL forward primer, 1 μL reverse primer, 1 μL probe and 4.6 μL of nuclease-free water to yield a 20 μL final reaction volume. Amplification reactions were performed in the QuantStudio™ 7 Flex Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) and programmed as follows: 1 cycle at 45 °C for 15 min, 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Virus RNA copies were calculated by generating a standard curve using a recombinant plasmid-containing virus segment insertion.