Primer Design and Plasmid Standard Preparations for Real-Time PCR (qPCR)

Highly conserved regions of TYLCV (GenBank no. KM435327.1) and ToCV (GenBank no. KC709510.1) were selected for the absolute quantification of TYLCV and ToCV, respectively, using the qPCR design tools from Integrated DNA Technologies Inc. (Coralville, IA, United States). The sequence and nucleotide coordinates of primers are provided in Supplementary Table 1. To obtain the insertion fragment for ToCV plasmid standards, previously obtained ToCV cDNA was amplified using the primer pair ToCV-qS3/ToCV-qA3, which produces a fragment of 157 bp, The PCR conditions were as follows: 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 20 s, with a final extension at 72°C for 7 min. After purifying the amplified fragments, they were cloned into the pMDTM 18-T vector (Takara Biotechnology Dalian Co., Ltd., China) and transformed into Escherichia coli DH5α competent cells. The PCR product’s insertion was verified by PCR screening and sequencing. Then, plasmid extractions were performed using a TIANpure Mini Plasmid Kit (Tiangen Biotech Beijing Co., Ltd.). The purity and concentration were measured using a Nano Photometer N60 (Implen Scientific Inc., Germany). A dilution series of 3.15 × 108 to 3.15 × 103 copies per μL was made for standard samples to develop a standard curve for the absolute quantification of ToCV genomic mRNA copies. The molecular copies of the standard samples were calculated using the following the formula (Shirima et al., 2017):

where NW represents the plasmid molecular weight expressed as plasmid size (bp) × molar mass per base (650 g mol–1 bp–1). The plasmid amount was calculated from the plasmid concentration determined using a Nano Photometer N60.

The construction of the recombinant TYLCV plasmid for standards was accomplished with the same procedure used for ToCV. The insertion fragment for TYLCV was amplified using the primer pair TYLCV-F/TYLCV-R, which produced a fragment of 194 bp. The PCR conditions were as follows: 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. A dilution series from 2.56 × 108 to 2.56 × 103 copies per μL of TYLCV plasmid was made for standard samples to develop a standard curve for the absolute quantification of TYLCV genomic DNA copies.

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