Optimized JEV RT-qPCR Diagnostic Assay

An in-house JEV RT-qPCR assay targeting the NS2A region, previously developed and validated [5 (link)], was used. JEV NS2A RT-qPCR was performed in duplicate using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Superscript-III kit; Thermo Fisher): 25-μL reaction volume; 600-nM forward (5’-AGCTGGGCCTTCTGGT-3’) and reverse (5’-CCCAAGCATCAGCACAAG-3’) primers; 300-nM probe (5’ FAM-CTTCGCAAGAGGTGGACGGCCA-TAMRA 3’) and 7.5 μL of RNA. Thermocycling conditions: 50°C for 15 minutes, 95°C for 2 minutes, 45 × [95°C for 15 seconds + 62°C for 45 seconds]. Positive (JEV RNA Genotype 3, UVE/JEV/UNK/TW/RP9-190 [GenBank KF907505, EVA 001V-02344]) and negative (no template) controls were performed for each RT-qPCR run. An internal control (10-μL MS2 phage) was added to each patient sample before extraction. Subsequently, a separate MS2 RT-qPCR run was performed to control the extraction process and to exclude inhibition, as previously described [21 (link)]. All RT-qPCR assays for optimization and subsequent experiments were run using a CFX96 qPCR detection system (Biorad Laboratories) with manual baseline and threshold settings. Cq >40 were reported as negative, and ≤40 as positive. The LOD was defined as the last dilution in which all the replicates were positive.