2.6. Full-Length Sequence of the Shanghai FcTTV Isolates

According to the FcTTV genome sequence (accession number {"type":"entrez-nucleotide","attrs":{"text":"HM142588","term_id":"299835119","term_text":"HM142588"}}HM142588) registered in GenBank, four primer pairs (Table 1) were designed to amplify the full-length sequences, which were obtained through nested PCR amplification, Sanger sequencing, and sequence assembly. Primers FcTTV-P1Q and FcTTV-P2Q were used for the first round of amplification and FcTTV-P3Q and FcTTV-P4Q were used for the second round of amplification.

Another sequence was amplified using primers FcTTV-P5Q and FcTTV-P6Q for the first round of amplification and FcTTV-P1 and FcTTV-P2 for the second round of amplification. Amplification was completed using Phanta Max Master mix (Vazyme Biotech Co., Ltd.).

The PCR positive samples were presented with a band at 1528 and 579 base pairs. PCR products were purified by the MinElute PCR Purification Kit 28004 (Qiagen, Hilden, Germany), ligated into the pMD20-T Vector (Takara Biomedical Technology (Beijing) Co., Ltd.), and cloned according to the manufacturer’s instructions. The clone products were sent to Sangon Biotech (Shanghai) Co., Ltd., Shanghai City, China, for sequencing.