Quantitative DENV RT-PCR analysis

For detection of DENV genome copies, one tenth of the volume of the homogenised sample was removed and added to an extraction buffer solution (10 mM Tris pH 8.2, 1 mM EDTA, 50 mM NaCl and proteinase K [33]) in a 1:1 ratio. Samples in extraction solution were incubated in a thermal cycler at 56°C for 5 min followed by 95°C for 5min [33] and then cooled on ice until use. DENV genome copies were determined by a 1-step quantitative reverse-transcriptase PCR (qRT-PCR) using TaqMan Fast Virus 1-Step Master Mix (ThermoFisher Scientific). Reactions contained 2.5 μl of sample, 4 × master mix, 250 μM forward primer, 250 μM reverse primer and 250 μM TaqMan FAM hydrolysis probe in a total 10 μl reaction volume. TaqMan primers and probes complementary to the 3’ untranslated region of DENV and the creation of the DENV-2 standard curve are described elsewhere [17,34]. Thermocycling conditions were as recommended by the manufacturer. Percentages of individuals infected with DENV were calculated; the lower detection limit for DENV-positive individuals was determined by detection of standards and set at 100 copies per qRT-PCR reaction.