T100 pcr cycler

The ddPCR was performed on a QX200 Droplet PCR platform (Bio-Rad, Pleasanton, CA, USA). ddPCR reaction was prepared with a final volume of 20 µL, which was comprised of 10 µL of 2× ddPCR Supermix (Bio-Rad, Pleasanton, CA, USA), 1 µL forward primer (10 μM), 1 µL reverse primer (10 μM), 0.4 µL probe (10 μM), 1 µL DNA template, and 6.6 µL ddH₂O. After the 20 μL reaction mix was prepared, the mixture was transferred to 8-well cartridges for emulsion droplets generation using a QX200 droplet generator (Bio-Rad, Pleasanton, CA, USA). Then, the generated water-in-oil droplets were transferred to a 96-well plate for amplification with a T100 PCR cycler (Bio-Rad, Pleasanton, CA, USA). The program for ddPCR amplification was: 95 °C for 5 min; 40 cycles of 30 s at 95 °C and 60 s at 58 °C. After thermal cycling, 98 °C for 10 min and then cooled to 4 °C. After PCR amplification, the 96-well plate was transferred to a QX200 droplet reader (Bio-Rad, Pleasanton, CA, USA) for fluorescent signal monitoring. The readout data was analyzed using QuantaSoft (Bio-Rad, Pleasanton, CA, USA).

To confirm the NGS result and to understand the differences between CiCV1 isolates from different host plants, we cloned and sequenced the complete nucleotide sequence of CiCV1-CN isolated in C. morifolium from Zhejiang Province, China. To obtain the full-length genome sequence of CiCV1-CN, a rapid amplification of cDNA ends (RACE) technique was utilized [6 (link),40 (link)]. We synthesized 5′- and 3′-RACE cDNAs using a SMARTer^® RACE 5′/3′ Kit (Takara Bio Inc., Dalian, China), following the manufacturer′s protocol. PCR amplification reactions were performed on a T100 PCR cycler (Bio-Rad, Pleasanton, CA, USA) using the KOD-plus DNA polymerase (Toyobo, Osaka, Japan). The primers used for the genome sequence cloning are listed in Supplementary Table S1. The obtained full-length genome sequence of CiCV1-CN was submitted to the NCBI Genbank database (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 7 February 2023) under an accession number OQ410649.