Arbovirus Detection in Mosquito Pools by PCR

Five mosquito species, consisting of one pool of Aedes albopictus, one pool of Armigeres subalbatus, one pool of Culex hutchinsoni, two pools of Culex tritaeniorhynchus, and five pools of Culex quinquefasciatus, were examined for arbovirus detection using polymerase chain reaction (PCR), following the procedure of Supriyono et al. [16 (link)] with PCR reagent modifications. RNA was extracted using tissue total RNA mini kit (Geneaid Biotech^®, Taiwan), according to the manufacturer’s recommendations. The total volume of PCR was 25 μL, consisting of 2.5 μL RNA sample, 12.5 μL of 2× MyTaq One-Step Mix, 1 μL of each forward and reverse primer (10 pmol/μL), 0.25 μL RT enzyme, 0.5 μL RiboSafe RNase inhibitor, and 7.5 μL diethyl pyrocarbonate-H₂O (Bioline, UK).
The primers used to amplify the sequences were MAMD and cFD2 with the non-structural protein target gene NS5 Flavivirus (252–260 bp long) and VIR2052F and VIR2052R with the non-structural protein target gene NS4 Alphavirus (144 bp long). Amplification was carried out using a SimpliAmp Thermal Cycler machine (Thermo Fisher Scientific, USA). The target gene, primer name, sequence, target size, and PCR conditions are presented in Table-1 [16 (link)]. The PCR product was electrophoresed on 1% agarose gel and visualized using an ultraviolet transilluminator.