Establishment Of A SYBR Green? Real-time Quantitative PCR For Detection Of Porcine Circovirus Type 3 And Its Molecular Epidemiology

Porcine circovirus type 3(PCV3)was newly proposed as a new porcine circovirus,which is first detected by American researchers in tissues of sows affected by reproductive failure in 2016.The virus is composed of 2.0 kb and the genome cording for capsid protein(Cap protein)contains 645 nucleotides,sharing 58.2%to 59.6%of nucleotide identities with the porcine circovirus type 2(PCV2).Clinical studies revealed that PCV3 is possibly associated with porcine dermatitis and nephropathy syndrome(PNDS),reproductive failure and cardiac and multisystemic inflammation.Therefore,it is urgent to conduct molecular epidemiological research on PCV3,which plays significant role in controlling disease.In this study,a pair of specific primers based on the conserved region of the ORF2gene sequence of PCV3 virus was designed and a PCV3 SYBR Green I fluorescence quantitative PCR detection method was established.The specific experimental results proved that the specificity of the detection method was high,and there was no cross-amplification curve with other common porcine viral genomes(such as PCV2,CSFV,PRV,PRRSV,PPV,etc.),and the dissolution curve peak was single;the sensitivity experiment results showed that the minimum detection amount of the method was 10~1copies/?L;and the coefficient of variation between the results of the inter-and intra-group reproducibility experiments was less than 3%;the detection of clinical samples verified that the method is suitable for clinical diagnosis Applicability,it can be applied to the clinical detection and diagnosis of PCV3.It's reported that there were infections both with no clinical signs and a range of clinical signs of abortion and reproductive failure,which makes infection with PCV3complicated.Although it's has been detected in China for a long time,epidemiological data on PCV3 are still lacking in nursery pigs and suckling pigs that are most susceptible to pathogens in clinical practice.In order to address the epidemiological characteristics of PCV3 in nursery pigs and suckling pigs and evolutionary history of this novel circovirus,molecular epidemiological investigation of PCV3 was conducted by PCV3 SYBR Green I fluorescence quantitative PCR detection 335 clinical swine samples collected from Guangdong,Fujian,Jiangxi,Guizhou and Anhui from 2016 to 2018 were screened for the presence of PCV3 by PCR detection 57 samples were positive for PCV3 and the positive rate was 17%.Serological positivity rate was 10.8%(4/37);the positive rate among nursery pigs was 22.7%.The positive rate among suckling pigs is 14.8%.Further analyses showed that the PCV3-positive rate at the farm level was 67.9%(19/28).23 full-genome sequences and 6 ORF2 gene sequences were obtained by PCR detection.Gene homology analysis showed that the sequences obtained in this experiment were 97.4%to 99.8%and 98.9%to99.9%homologous to published standard reference sequences in China and abroad,respectively.The amino acid sequence comparison analysis of ORF2 gene revealed that the Cap protein of PCV3 mainly contains 4 amino acid mutation regions at position 5~10,20~27,98~104 and 146~150.Genome-based phylogenetic tree analysis showed that PCV3was distinct from PCV2 concerning evolution dynamics,belonging to different branches of phylogenetic tree.Evolutionary analyses revealed that the origin of PCV3 in China is complex,sharing high identical with the strains from varies areas in the world.In this study,we successfully established an easy use,high-sensitivity and low-cost fluorescent quantitative PCR assay for the accurate diagnosis of PCV3 disease.Also the PCV3 epidemiological investigation results laid a theoretical foundation for the research of PCV3 and the prevention of PCV3 for pig farms.