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

<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 10⁻³ 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.</p

Evaluation of the three optimised RT-qPCR assays using patient samples with Central Nervous System (CNS) infections.

<p>Patients with RT-qPCR ‘positive’ test results.</p

Primers and probes evaluated during the study.

<p>Primers and probes evaluated during the study.</p

Cq results for validation of the optimised JEV RT-qPCR assays.

<p>Cq results for validation of the optimised JEV RT-qPCR assays.</p

Standard reaction mixes and cycling conditions.

<p>Standard reaction mixes and cycling conditions.</p

Anti-JEV IgM ELISA (JEV MAC-ELISA) results of consecutive patient samples comparing Dried Cerebrospinal Fluid Spots (DCS) on filter paper using the pre-cut protocol vs routine neat Cerebrospinal Fluid.

<p>Anti-JEV IgM ELISA (JEV MAC-ELISA) results of consecutive patient samples comparing Dried Cerebrospinal Fluid Spots (DCS) on filter paper using the pre-cut protocol vs routine neat Cerebrospinal Fluid.</p

Study sites: Geographical distribution of four hospitals involved in patient recruitment, in Vientiane (colour map), and with respect to the Lao PDR (grey map).

<p>Study sites: Geographical distribution of four hospitals involved in patient recruitment, in Vientiane (colour map), and with respect to the Lao PDR (grey map).</p

Comparisons of the prevalence of <i>T</i>. <i>whipplei</i> carriage in children feces between Senegal and Laos.

<p>Comparisons of the prevalence of <i>T</i>. <i>whipplei</i> carriage in children feces between Senegal and Laos.</p

Optimisation experiments performed for the three best performing RT-qPCR systems^*.

<p>Optimisation experiments performed for the three best performing RT-qPCR systems^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194412#t003fn001" target="_blank">*</a>}.</p

JEV MAC-ELISA results from DCS using CSF-substitutes, 903 filter paper and various protocols for sample spotting.

<p>JEV MAC-ELISA results from DCS using CSF-substitutes, 903 filter paper and various protocols for sample spotting.</p