Establishment And Preliminary Application Of PCV3 SYBR Green Ⅰ Fluorescent Quantitative PCR Assay

In 2015,a new type of porcine circovirus PCV3 was discovered at a commercial pig farm in North Carolina in the United States following the outbreak of porcine reproductive disorders and porcine dermatitis nephrotic syndrome(PDNS).In China,South Korea,Poland,Denmark,Spain,Italy and Brazil,PCV3 infection cases have been reported one after another henceforth.Domestic studies have found that cases of PCV3 infection have been reported in Guangdong,Guangxi,Shandong,Hunan,Hubei,Liaoning and Jiangsu provinces and regions.Furthermore,synthesizing existing PCV3 research,it is reported that PCV3 may be related to sow reproductive disorders,PDNS,multisystem inflammation,myocarditis and tremor of newborn piglets.The widespread infection of PCV2 in pig farms in China has caused a huge economic loss to the pig industry.The detection of PCV3 in PDNS sows and aborted fetuses needs to be highly regarded by the domestic industry.Therefore,it is of great significance to establish a rapid,simple,specific and sensitive PCV3 detection system and explore its detection effectiveness.In this study,nested PCR was used to detect PCV3 in porcine lung tissue,and to amplify the full-length sequence of cap gene in PCV3 positive sample,and TA cloning technique was used to obtain and analyze the sequence information.The amino acid sequence alignment analysis of PCV3 and PCV2 cap showed that PCV3 cap did not possess PXXP motif,did not contain cysteine cysteine,and lacked 8 amino acids in the Loop CD region.Based on the sequence of PCV3 cap gene,specific primers were designed,and the SYBR Green Ⅰ fluorescence quantitative PCR detection technology system was established.The detection limit was 102copies/uL,the detection efficiency(E)was 94.2%,and the regression squared value(R2)was 0.998.The coefficient of variation within and between batches was less than 2%,and for other common swine viruses(PCV2,PRV,PPV,PRRSV,PEDV,PCV1 and CSFV)were insensitive and highly PCV3 specific.Using PCV3 SYBR Green Ⅰ fluorescence quantitative PCR detection technology,190 semen samples,from January 2015 to December 2016,were collected(85 for serum of sow with abortion or reproductive records,105 healthy serum),and DNA of 75 semen samples was detected with PCV3,the result showed that the positive rate of PCV3 was 32.63%(sow serum)and 4.0%(semen)respectively.6 PCV3 cap full-length sequences(GenBank MG745690-MG745695)were obtained from serum samples of PCV3 positive sows.Sequence analysis showed that these 6 sequences had 96% to 99.7% nucleotide homology with 51 PCV3 complete genome sequences reported by GenBank.By analyzing the genetic evolution of cap gene of PCV3 strain,it was found that PCV3 was different from PCV1 and PCV2 in evolutionary branch,and the six sequences belonged to PCV3 a branch,while 3a and American strain were in the same evolutionary branch.A new type of porcine circovirus PCV3 was detected in serum of sows and semen samples collected in Hunan province.Therefore,a comprehensive investigation of PCV3 infection is still needed.Based on the PCV3 cap sequence,a fluorescent quantitative detection technology system for PCV3 SYBR Green Ⅰ was successfully established,which laid an experimental foundation for the clinical diagnosis of PCV3 and the evaluation of vaccine potency in the future.The PCV3 cap sequence and its mutation information from the serum of reproductive disorder sows provide a reference experimental basis for the study of PCV3 pathogenicity and vaccine design.