High pure nucleic acid kit

The detection of viral RNA in each individual was done by qRT-PCR [13 (link)]. Mosquitoes were kept under laboratory conditions. At three time points—3, 7 and 14 days after intrathoracic infection (d.p.i.)—they were individually stored in tubes and cryopreserved in a −80°C freezer for the preservation of viral RNA. After the collection and storing of all mosquitoes in all doses and time points, Ae. aegypti were individually assayed for viral load.
For RNA extraction each mosquito was, separately, put into a 2.0mL vial-tube with one glass bead 2mm and 50 μL of PBS Buffer one-fold. The mosquitoes were then beaten for 90 seconds on Bead-beater machine (Biospec Products). After that, we performed the RNA extraction using High Pure Nucleic Acid kit (Roche) commercial kit following the manufacturer instructions. The RNAs were then quantified on Nanodrop Spectrophotometer (Thermo Scientific) and diluted to 50ng/μL. For virus detection, we used a pair of primers DENV-Forw: 5’-AAG GAC TAG AGG TTA GAG GAG ACC C- 3’ and DENV-Rev: 5’- CGT TCT GTG CCT GGA ATG ATG- 3’ and DENV: 5’-HEX/AAC AGC ATA TTG ACG CTG GGA GAG ACC AGA/3BHQ_1/3’ probe that amplifies a 109pb fragment.

According to the manufacturer’s instructions, DNA and RNA from each sample (stool and sera pools and tissues) were extracted using the High pure nucleic acid kit (Roche^®, Ref 11858874001, Mannheim, Germany). PCV2, PCV3, and PCV4 were detected by conventional PCR. It is important to note that samples from period A were initially evaluated for PEDV in 2016. Primers for PCV2, PCV3, and PCV4 are presented in Table 4. In brief, reactions were performed in a total volume of 25 µL containing 0.25 µL of Taq polymerase (5 U/µL) (Go taq flexi-Promega^®, Ref M8295, Madison, WI, USA), 5X Taq buffer (2.5 µL), 2 mM MgCl2, 0.5 mM dNTPs, 1 µL of each primer (20 µM), and 2 µL of extracted DNA. The PCR reactions were performed on a Biorad^®-DNA (Hercules, CA, USA) thermocycler using a protocol consisting of denaturation at 94 °C for 5 min, followed by 35 cycles including a denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 5 min. Positive/negative status for PCV2, PCV3, and PCV4 was determined by the presence of 505, 340, and 391 bp bands, respectively, on 1% agarose gel.