Development and Application of A SYBR Green Ⅰ Real-time Fluorescent Quantitative RT-PCR Method for Detection of Swine Sapporo Virus,Along with Analysis of the Virus Variation and Evolution
The porcine sapovirus(PoSaV)is classified as an enterovirus and is known to induce acute gastroenteritis in pigs,presenting a significant threat to the swine breeding industry.The PoSaV-infected pigs exhibited symptoms similar to those caused by other diarrheal viruses and were difficult to distinguish.We designed a PoSaV assay to detect the virus and assess its prevalence in pig farms.According to the GⅢgenotype in GenBank,primers were designed,the reaction conditions were optimized,and the fluorescence quantitative RT-PCR method was established by specificity evaluation,sensitivity,and repeatability detection.In this study,20 anal swab samples were collected from diarrhea sows in Guizhou province and detected by RT-PCR.The whole VP1 gene was sequenced.MEGA 7.0 was used for phylogenetic tree construction and homology analysis.The results showed that the established fluorescence quantitative RT-PCR detection method had high specificity and sensitivity.At a viral copy number of 1× 101·μL-1 the method showed excellent repeatability and stability.Both intra-assay and inter-assay coefficients of variation were less than 1％.The positive rate of porcine diarrhea in clinical samples was determined to be 85％(17/20)during testing.The 17 VP1 gene sequences obtained were compared with 14 reference strains in China and abroad.The results revealed that the nucleotide homology between all sequenced strains and the reference sequence ranged from 81.0％to 99.2％,while the amino acid homology ranged from 80.8％to 97.6％.Furthermore,it was observed that all strains were significantly different from the classic Cowden strain.All the sequenced strains showed close genetic similarity to YNAN and YNAN2020,with five of them(GZ-VP1-3,GZ-VP1-5,GZ-VP1-6,GZ-VP1-9,GZ-VP1-11)sharing 99.2％and 96.9-97.6％in nucleotide and amino acid homology with the YNAN strain in Yunnan province.This suggests that they may have evolved from an original strain.All sequenced strains were clustered closely on the same branch,with nucleotide and amino acid homology ranging from 91.8％to 100％,indicating a certain degree of variation in the VP1 gene.This study successfully established a detection method for PoSaV and analyzed the genetic variation of current PoSaV strains,which provide valuable insights for prevention and control of PoSaV.
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