猪捷申病毒基因4型云南流行株的分离与鉴定OA

This study aimed to establish an efficient and accurate real-time fluorescent quantitative PCR assay for detection of porcine Te-schovirus(PTV),and to isolate and identify representative epidemic strains in Yunnan Province.Primers were designed in the conserved re-gion of the 5'-UTR non-coding region by aligning the complete PTV genomes deposited in GenBank in recent years,with degenerate bases introduced,and a SYBR Green Ⅰ fluorescent quantitative PCR method was established.Subsequently,the positive sample of the Yunnan ep-idemic strain PTV-4 was inoculated into porcine kidney cells(PK-15)for virus isolation and determination of its biological characteristics.The results were as follows:The standard curve of the established fluorescent quantitative PCR method showed a favorable linear relationship with a correlation coefficient R2 of 0.993 4,and the minimum detection limit was 8.35×104 copies/μL.Both the intra-assay and inter-assay coefficients of variation were less than 3%,and no cross-reaction was observed with other pathogens,indicating excellent specificity.After inoculation of PK-15 cells with the PTV-4 positive sample,cytopathic effects began to appear at 24 hours post-inoculation,mainly charac-terized by cell shrinkage,granulation and aggregated adherence,followed by cell detachment after 72 hours.When passaged to the 11th gen-eration,the viral load increased from 8.78×104 copies/μL to 8.16×107 copies/μL,demonstrating stable proliferation capacity.The trans-mission electron microscopy revealed spherical,non-enveloped virions with a diameter of approximately 26-30 nm.In conclusion,this study established a SYBR Green Ⅰ fluorescent quantitative PCR detection method applicable to clinical diagnosis and epidemiological investigation.The obtained Yunnan epidemic strain of PTV laid a foundation for subsequent research on its structural function,pathogenic mechanism,im-mune prevention and other related aspects.