Zika Virus Detection in Mosquitoes and Urine

Individual mosquitoes were placed in 1.5 ml microcentrifuge tubes with 200 µl of 1X phosphate buffered saline (PBS), ground, and centrifuged at 13,000×g for 10 min. Then, 200 µl of the supernatant was transferred to a sterile 1.5 ml microcentrifuge tube mixed with 200 µl of 2X minimum essential medium Eagle (MEM) (HyClone, USA) containing 2% heat-inactivated FBS (Gibco, USA), 2% penicillin (100 U/ml), and streptomycin (100 µg/ml) (Sigma-Aldrich, USA), and stored at − 80 °C for further virus isolation attempts. The mosquito carcass or human urine sample was mixed with 300 µl of lysis buffer and processed for viral RNA extraction using an Invisorb Spin Virus RNA Mini Kit (STRATEC Molecular GmbH, Germany). The mosquito or human urine RNA samples were amplified for detection of ZIKV at NS5 gene using hn-RT-PCR (Thai Patent No. 2001004011, 2020)^{15 (link)}. The positive control was constructed and used the lower band of a synthetic positive control plasmid. The amplified PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized using Quantity One Quantification Analysis Software Version 4.5.2, Gel Doc EQ System (Bio-Rad, USA).