63 research outputs found

    Development of an improved RT-qPCR Assay for detection of <i>Japanese encephalitis virus</i> (JEV) RNA including a systematic review and comprehensive comparison with published methods - Fig 1

    No full text
    <p>Standard curves of the 1) Pyke, 2) NS2A assays and 3) NS3 assays with G3-RP9-190 on (A) Day 1 and repeated on (B) Day 2. Result of the RT-qPCR run, ‘Cq’, is plotted against the ‘log starting copies number’, at the RNA dilutions detected: Pyke assay 1:10 serial dilutions of G1-769 in triplicate at 10<sup>−3</sup> to 10<sup>−6</sup>; NS2A assay 1:10 serial dilutions of G1-769 in triplicate at 10<sup>−3</sup> to 10<sup>−7</sup>; and NS3 with G3-RP-190 10<sup>−4</sup> to 10<sup>−7</sup>. Efficiency = 10<sup>−1/slope</sup>-1. R<sup>2</sup> = Correlation Coefficient. RT-qPCR performed with Fastvirus kit (TaqMan® Fast Virus 1-Step) with a reaction volume of 50μL, sample volume of 30μl, and primer and probe concentrations of 600nM and 300nM respectively. Thermocycling conditions were 50°C for 5 minutes, 95°C for 20 seconds and 45 x (95°C for 15 seconds + x°C for 60 seconds). The optimal annealing temperature ‘x°C’ was different for each assay: 62°C, 60°C and 56°C for the Pyke, NS2A and NS3 assays respectively.</p
    corecore