Complete genome sequence of a Rodent Torque teno virus in Hainan Island, China and establishment of a real-time PCR for detecting Rodent Torque teno virus 3

摘要

Objective: To perform whole-genome sequencing and phylogenetic analysis of the localendemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattusnorvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCRdetection assay for RoTTV3. Methods: Based on the high-throughput genome sequencinganalysis, specific primers were designed and the whole genome sequence was amplifiedby PCR and Sanger sequencing. Specific detection primers were designed based on theconserved sequences of RoTTV3. The recombinant plasmid contained the whole genome ofRoTTV3-HMU1 was constructed as a standard control. The experimental conditions wereoptimized and the real-time PCR detection assay of RoTTV3 was established. Results: Thegenomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfullysequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype.In this experiment, the real-time PCR detection method of RoTTV3 was established. Thestandard curve generated had a wide dynamic range from 1伊(102-108) copies/μL, with a linearcorrelation (R2=1.000). The melting curve analysis using SYBR Green showed only onespecific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction. Discussion: This study was the first report of the RoTTV in Hainan Islands, and itsphylogenetic analysis was of great significance to the origin and evolution of RoTTV. TheRoTTV3 real-time PCR detection method established in this experiment has a high sensitivityand good specificity, which lays a technical foundation for the epidemiological investigation ofRoTTV3.