Design of gBlocks and qPCR primers

Based on the alignment result, one genomic region of 999 nt containing all binding sites of the LAMP primers was selected. Three 999-nt double-stranded DNA fragments, corresponding to the RNA sequence of this genomic region in one selected ToBRFV strain (gBlock^ToBRFV; INSDC no: {"type":"entrez-nucleotide","attrs":{"text":"KT383474","term_id":"954622602","term_text":"KT383474"}}KT383474; nt 1657–2655), one selected TMV strain (gBlock^TMV; INSDC no: {"type":"entrez-nucleotide","attrs":{"text":"FR878069","term_id":"371786069","term_text":"FR878069"}}FR878069; nt 1649–2647) and one selected ToMV strain (gBlock^ToMV; INSDC no: {"type":"entrez-nucleotide","attrs":{"text":"MH507166","term_id":"1450640870","term_text":"MH507166"}}MH507166; nt 1653–2651) were synthesized as gBlocks. In addition, two gBlocks, in which the sequences of the binding site of primer F1c or B2 on gBlock^ToBRFV were replaced with the corresponding sequences in one of the ToMV strains ({"type":"entrez-nucleotide","attrs":{"text":"DQ873692","term_id":"115607483","term_text":"DQ873692"}}DQ873692), were synthesized and named gBlock^ΔF1c and gBlock^ΔB2, respectively (Table 1 and Fig 1). Based on the sequence of gBlock^ToBRFV, a pair of qPCR primers was designed using the Primer3 software (http://primer3.ut.ee). This primer pair was name qF1/qR1 (Table 1).