2.18. ZIKV RNA Detection by Polymerase Chain Reaction with Reverse Transcriptase (RT-PCR)

The ZIKV RNA detection in a single step by the RT-PCR reaction has been previously described [39,40]. The master mix was prepared according to the specifications given by the OneStep RT-PCR kit (Qiagen). The primers ZIKV FW [5′-GCTGGDGCRGACACHGGRACT-3′] (Mfg. ID 275853243, Integrated DNA Technologies [IDT], San Diego, CA, USA)] and ZIKV RV [5′-RTCYACYGCCATYTGGRCTG-3′] (Mfg. ID 275853246, IDT, USA)] developed by Faye et al. [40] were used. The reaction was performed on a GeneAmp PCR System 2400 (Applied Biosystems, Foster City, CA, USA) with the following conditions: pre-PCR at 50 °C for 40 min and 95 °C for 15 min; 35 cycles at 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 1 min; final elongation at 72 °C for 7 min. The amplified cDNA (amplicon of 364 bp) corresponding to the more specific genome region for the ZIKV E protein, which has no cross reaction with other Flavivirus [40], was visualized in 2% ethidium bromide-stained 1.2% agarose gel using a Typhoon FLA 9500 scanner with GE control software. The images were analyzed with ImageJ software.