Superscript 3 platinum one step rt qpcr system kit

The SuperScript III Platinum One-step RT-qPCR System kit (Invitrogen, California, USA) was used in the 7500 Fast Real-Time PCR System. Specific primers and probes were used for each of four DENV serotypes, and the primers and probe of the human RNase P (RP) gene used as an internal control (S1 Table). The serotypes were evaluated in a multiplex reaction with the following reaction mixture: 12.5 μL of 2x Reaction Mix, 0.25 μL of each of the primers D1 and D3, 0.125 μL of each of the primers D2 and D4, 0.045 μL of each probe, 0.5 μL of enzyme, 5.32 μL of RNase-free water, and 5 μL of RNA. The thermocycling conditions were 1 cycle of 50°C for 30 min, followed by 1 cycle of 95°C for 10 min, 45 cycles of 95°C for 15 s and 60°C for 1 min [16 (link)].

The SuperScript™ III Platinum^® One-Step RT-qPCR System Kit (Invitrogen, Carlsbad, Califórnia, EUA. Catalog: 12574035) was used in a 7500 Fast Real-Time PCR System (Applied Biosystems^®, Foster City, Califórnia, EUA) to amplify viral genetic material. Primers and probes were used for each of the following viruses: HMPV, HRSV, HPIV 1–4, betacoronavirus 1 (βCoV1), human coronavirus (HKU1, 229E, NL63) (HCoV), HMdV, RV, EV, and PBpV. The human RNAse P (RP) gene was used as an internal control (Table 1). The viruses were evaluated in uniplex reactions with the following reaction mixture: 12.5 μL of 2x Reaction Mix, 0.5 μL of each primer and probe, 1 μL of enzyme, 5.5 μL of RNAse-free water, and 5 μL of total nucleic acid. The following thermocycling conditions were used: one cycle of 45°C for 10 min and 95°C for 10 min and 45 cycles of 95°C for 15 sec and 55°C for 1 min.

RNA samples were extracted directly from cultured cells using Trizol reagent (Invitrogen, Waltham, MA, USA) followed by isopropanol precipitation, and sample quality control was performed with Agilent Bioanalyzer 2100. All of the samples had an RNA integrity number ranging from 9.9 to 10. Briefly, mRNA quantitation was performed by RT-qPCR using the Superscript III platinum One-step RT-qPCR System kit (Invitrogen, Waltham, MA, USA) and normalized on Actinb mRNA. Oligonucleotide sequences are indicated in Table S2.