Superscript 3 platinum one step quantitative kit

RNA of the A/Bretagne/7608/2009 (A(H1N1)pdm09) and A/Centre/1003/2012 (A(H3N2)) influenza virus mixes was extracted from culture supernatants using the Easy Mag platform (BioMérieux, #280130-#280134 and #280146) according to the manufacturer’s instructions. Extraction of nucleic acids of clinical specimens was performed using the Viral RNA/DNA isolation kit (Macherey Nagel, Germany, cat No: MN 740691.4). The RNA extraction was done according to manufacturer’s instructions except that the beads were not washed in buffer MV5 but instead left to dry for 10 minutes until the pellet did not appear shiny anymore.
Using 5 µl RNA for each sample, a RT-qPCR was performed using the SuperScriptIII Platinum One-Step Quantitative Kit (Invitrogen) with primers InfA_Forward, InfA_Reverse and InfA_probe. These bind to an influenza M gene section [41 ]. Each reaction contained 0.5 µl primer/probe, 1 µl SuperScript III RT/Platinum Taq mix, 5 µl nuclease-free water, 12.5 µl PCR Master Mix and 5 µl RNA.

RNA was extracted from samples using the QIAamp Viral RNA Mini Kit (Qiagen), according to the manufacturer's instructions. RNA quantity and purity were evaluated on the basis of absorbance (A₂₆₀/A₂₈₀ ratio) using NanoDrop ND‐1000 (Thermo Fisher), and the integrity of RNA extracted was verified using RNaseP (human RNase P gene) primer and probe as reverse transcription–polymerase chain reaction internal controls. Following the standard CDC protocol,20 each sample was subjected to reverse transcription–real‐time polymerase chain reaction by using primers and probe sets specific for detection of influenza A(H1N1)pdm09 and influenza A/H3. All internal positive and negative controls were included on reactions, as described on the cited protocol. Briefly, reactions using SuperScript III Platinum One‐Step Quantitative Kit (catalogue no. 11745‐100, Invitrogen) containing 0.5 μL of SSIII Platinum Taq Mix, 1μM of each primer, 0.25μM of probe, 12.5 μL of 2X Master Mix, 5 μL of RNA sample, and water to a final volume of 20 μL were performed in an Applied Biosystems 7500 Real‐Time PCR System (Thermo Fisher) following 50°C for 30 minutes, 95°C for 2 minutes, and 45 cycles at 95°C for 15 seconds and 55°C for 35 seconds.

Viral loads were measured in bronchoalveolar lavage (BAL) samples by semi-quantitative real-time RT-PCR using the CDC protocol for swine influenza A (H1N1) (kit obtained from BEI Repository NR-15577) [79 (link)]. Briefly, RNA was extracted from BAL samples using a modified version of the Invitrogen PureLink Viral RNA/DNA kit (cat. # 12280–050). Semi-quantitative RT-PCR was performed using Invitrogen SuperScript III Platinum One-Step Quantitative Kit and Swine Influenza H1 (swH1) primers and probe from the BEI kit. A standard curve was generated by extracting RNA from 10-fold dilutions of CA09 virus of known chicken egg infectious dose 50 (CEID₅₀). The relative CEID₅₀ of unknown NHP samples were back calculated by comparison with CT values from this standard curve and are expressed as CEID₅₀ equivalents/ml. No template and positive (a sample from a prior experiment) template controls were included in each PCR run.