Superscript 3 platinum one step quantitative rt pcr system kit

A total of 140 μl of serum was used for RNA extraction with the RNeasy Mini Kit (Qiagen, Germany). Real-time RT-PCR was conducted with the SuperScript III Platinum One-Step Quantitative RT-PCR System Kit (Invitrogen, USA). We followed the kit instructions to conduct the experiments. Reaction parameters were 50 °C for 30 min, 95 °C for 2 min, and then 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The primers and probes located in the L, M, and S segments of SFTSV were from a previous study [10 (link)]. The cut-off cycle threshold (Ct) value was set at 35 cycles.

The quantification of the YFV genome from the total RNA extracted from the mosquito pools was performed using RT–qPCR [43 (link)] together with the standard curve prepared in the previous step. The reaction was performed using the Superscript III^® Platinum^® One-Step Quantitative RT–PCR System kit (Invitrogen) in a total volume of 25 μL, containing 12.5 nM of each primer (YfallF-GCT AAT TGA GGT GYA TTG GTC TGC and YfallR-CTG CTA ATC GCT CAA MGA ACG CAC), 5 nM of probe (YfallP-ATC GAG TTG CTA GGC AAT AAA, labeled with FAM and BHQ1) and 5 μL of the extracted total mosquito RNA. The runs were performed in a 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The cycling conditions were as follows: 50 °C for 30 min, followed by a 2-min cycle at 95 °C, then 45 cycles of 15 s at 95 °C and 1 min at 60 °C. Samples with CT values below 37 were considered positive. The YFV genome concentration in the samples was determined in RNA copies/µL using a standard curve in the Applied Biosystems 7500 Software version 2.3. The values were then converted into copies/mg of mosquito using the following formula: $$Q=\frac{\left(\frac{q\times 60\times 1000}{140}\right)}{10\times 1}$$
where,
Q = quantification in copies/mg,
q = quantification in copies/µL,
60 = RNA elution volume,
1000 = volume of PBS used in the maceration,
140 = volume of macerate used in the extraction,
10 = number of mosquitoes per pool,
1 = mean mass of 1 mosquito in mg.