Purelink viral rna mini kit

Total RNA was isolated using a PureLink viral RNA mini kit (Thermo Fisher Scientific, Waltham, MA, USA). Single-stranded cDNA was synthesized from RNA (1 μg) using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and was used for quantitative polymerase chain reaction (qPCR) with the specific primers and universal probe library probes presented in Additional file 1: Table S1. Target gene expression levels were calculated by the ΔΔCT method, with β-ACTIN serving as an internal control for normalization.

In all of the cases, viral RNAs were extracted from a starting volume of 200 μl of sera using the PureLink Viral RNA Mini Kit (Life Technologies, USA) and immediately subjected to real-time qRT-PCR analysis. Real-time qRT-PCR was performed with a Mx3000P machine (Stratagene, USA). The primer sequences applied for detecting and typing DENV and the formulation of the qRT-PCR have been described elsewhere [22 (link), 24 (link)]. Each run included negative controls spiked with water and positive controls spiked with DENV RNA extracted from culture fluid containing DENV. The criteria of a positive control were a threshold cycle (Ct) value of ≤30 and a Tm ≥79°C, while a negative control had a Ct value ≥40 and a Tm <79°C. For the samples, a Ct value of ≤30 or a Tm ≥79°C was considered positive.

For the detection of DENV genome in pooled sera, viral RNA was extracted from 200 μL of the pooled serum (combined ten single serum aliquots into one pool) or the control serum by using the PureLink Viral RNA Mini Kit (Life Technologies; USA) and immediately subjected to real-time RT-PCR analysis. Dengue-specific primers for the DENV RNA detection were DN-F: CAA TAT GCT GAA ACG CGA GAG AAA and DN-R: CCC CAT CTA TTC AGA ATC CCT GCT. Serotype-specific primers for molecular serotyping were DN-F: CAA TAT GCT GAA ACG CGA GAG AAA, D1-R CGC TCC ATA CAT CTT GAA TGA G, D2-R: AAG ACA TTG ATG GCT TTT GA, D3-R: AAG ACG TAA ATA GCC CCC GAC and D4-R: AGG ACT CGC AAA AAC GTG ATG AAT [18 (link)]. These primers amplified the genomic region encoding the nucleocapsid or core protein. Real-time RT-PCR was performed in a Mx3000P machine (Agilent/Stratagene, USA) by using the Brilliant II SYBR Green QRT-PCR Low ROX Master Mix system (Agilent/Stratagene, USA). Amplification plots and Tm values were analyzed to verify the specificity of the amplicon.

Viral RNA was extracted from 200 μL of serum sample using the PureLink Viral RNA Mini Kit (Life Technologies, USA) according to the instructions from the manufacturer and immediately subjected to real-time qRT‒PCR. Real-time qRT‒PCR was performed with a Brilliant II SYBR Green qRT‒PCR Low ROX Master Mix system (Agilent, USA). In brief, a 25 μL mixture containing 5 μL of sample RNA, 0.25 μM forward and reverse DENV detecting or molecular-typing primers each, 2 × Brilliant II SYBR Green qRT‒PCR Low ROX Master Mix, RT/RNase Block Enzyme Mixture and RNase-free water was assayed in an Mx3000P machine (Agilent, USA). Dengue-specific primers for DENV RNA detection were DN-F: CAA TAT GCT GAA ACG CGA GAG AAA and DN-R: CCC CAT CTA TTC AGA ATC CCT GCT. Serotype-specific primers for molecular serotyping were DN-F: CAA TAT GCT GAA ACG CGA GAG AAA, D1-R CGC TCC ATA CAT CTT GAA TGA G, D2-R: AAG ACA TTG ATG GCT TTT GA, D3-R: AAG ACG TAA ATA GCC CCC GAC and D4-R: AGG ACT CGC AAA AAC GTG ATG AAT. The criteria of a positive control were a threshold cycle (C_t) value ≤ 30 and a Tm ≥ 79 °C, while a negative control had a C_t value ≥ 40 and a Tm < 79 °C. For the samples, a Ct value of ≤ 30 or a Tm ≥ 79 °C was considered positive [17 (link)].

Viral RNAs were extracted from 200 μl of the cell culture supernatants or serum samples using the PureLink^® Viral RNA Mini Kit (Life Technologies; USA) according to the manufacturer’s instructions and stored at -80°C. Real-time qRT-PCR was performed using a Mx3000P quantitative PCR system (Agilent/Stratagene, USA). The samples were assayed in 25-μl reaction mixtures containing 5 μl of the sample RNA and 0.2 μM forward and reverse primers using the Brilliant II SYBR Green QRT-PCR Low ROX Master Mix and RT/RNase Block Enzyme Mixture (Agilent/Stratagene, USA). The primer sequences applied for detecting and typing the dengue viruses were published previously [10 (link)]. The thermal profile used for the one-step SYBR Green-based real-time qRT-PCR assay and the melting curve analysis were based on previously described information with minor modifications [10 (link)], and amplification plots and Tm values were routinely analyzed to verify the specificity of the amplicons.