Table 1.
Standard reaction mixes and cycling conditions.
Table 2.
Comparison of RT-qPCR conditions using the pre-existing in-house RT-qPCR assay*.
Table 3.
Optimisation experiments performed for the three best performing RT-qPCR systems*.
Table 4.
Primers and probes evaluated during the study.
Fig 1.
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 10−3 to 10−6; NS2A assay 1:10 serial dilutions of G1-769 in triplicate at 10−3 to 10−7; and NS3 with G3-RP-190 10−4 to 10−7. Efficiency = 10−1/slope-1. R2 = 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.
Table 5.
Cq results for validation of the optimised JEV RT-qPCR assays.
Table 6.
Evaluation of the three optimised RT-qPCR assays using patient samples with Central Nervous System (CNS) infections.
Patients with RT-qPCR ‘positive’ test results.