Primer and Probe Design

To design efficient primer pairs and probes, conserved regions of the viral genome were identified in JEV and GETV strains. The nucleotide sequence of the NS1 gene of JEV reference strain N28 was obtained from GenBank (no. {"type":"entrez-nucleotide","attrs":{"text":"MH753126","term_id":"1547148250","term_text":"MH753126"}}MH753126) and compared with the NS1 gene sequence of other JEV strains ({"type":"entrez-nucleotide","attrs":{"text":"JN381843","term_id":"347301214","term_text":"JN381843"}}JN381843, {"type":"entrez-nucleotide","attrs":{"text":"HM366552","term_id":"313105111","term_text":"HM366552"}}HM366552, {"type":"entrez-nucleotide","attrs":{"text":"KT957423","term_id":"1018193539","term_text":"KT957423"}}KT957423, {"type":"entrez-nucleotide","attrs":{"text":"KU508408","term_id":"1045961485","term_text":"KU508408"}}KU508408, {"type":"entrez-nucleotide","attrs":{"text":"KC183732","term_id":"490255889","term_text":"KC183732"}}KC183732, {"type":"entrez-nucleotide","attrs":{"text":"JN711458","term_id":"373221502","term_text":"JN711458"}}JN711458, {"type":"entrez-nucleotide","attrs":{"text":"AF075723","term_id":"3342805","term_text":"AF075723"}}AF075723, {"type":"entrez-nucleotide","attrs":{"text":"KX945367","term_id":"1180968678","term_text":"KX945367"}}KX945367, {"type":"entrez-nucleotide","attrs":{"text":"MN544779","term_id":"1827520457","term_text":"MN544779"}}MN544779). The nucleotide sequence of the E2 gene of GETV reference strain M1 was obtained from GenBank (no. {"type":"entrez-nucleotide","attrs":{"text":"EU015061","term_id":"157734228","term_text":"EU015061"}}EU015061) and compared with the E2 gene sequence of other GETV strains ({"type":"entrez-nucleotide","attrs":{"text":"EU015063","term_id":"157734234","term_text":"EU015063"}}EU015063, {"type":"entrez-nucleotide","attrs":{"text":"KY434327","term_id":"1186228355","term_text":"KY434327"}}KY434327, {"type":"entrez-nucleotide","attrs":{"text":"KY450683","term_id":"1248695964","term_text":"KY450683"}}KY450683, {"type":"entrez-nucleotide","attrs":{"text":"EU015062","term_id":"157734231","term_text":"EU015062"}}EU015062, {"type":"entrez-nucleotide","attrs":{"text":"MG869691","term_id":"1386679852","term_text":"MG869691"}}MG869691, {"type":"entrez-nucleotide","attrs":{"text":"EF631998","term_id":"149346282","term_text":"EF631998"}}EF631998). Nucleotide sequence data were retrieved from the GenBank database and CLUSTAL W algorithm implemented in the MegAlign of DNASTAR program package (MegAlign 5.00, DNASTAR Inc., Madison, USA).Oligonucleotide primers and probes used for JEV and GETV real-time amplification were designed using SnapGene software (SnapGene^®4.1.9) software against the non-structural gene (NS1) of JEV and the structural gene (E2) of GETV. Potential target regions were selected, and primers were synthesized and evaluated for use in quantitative real-time RT-PCR. The nucleotide sequences of the JEV- and GETV-specific primer pairs and probes, and the characteristics of the amplicons are shown in Table 1.

Oligonucleotide primers and fluorogenic probes used in the duplex TaqMan RT-qPCR assay.

Numbering based on {"type":"entrez-nucleotide","attrs":{"text":"MH753126","term_id":"1547148250","term_text":"MH753126"}}MH753126 (N28, JEV) and {"type":"entrez-nucleotide","attrs":{"text":"EU015061","term_id":"157734228","term_text":"EU015061"}}EU015061 (M1, GETV). V = A/G/C.