Isolation, Identification And Diagnosis Establishment Of Real-Time PCR For Porcine Circovirus Type 2

Porcine Circovirus(PCV) is a small icosahedral nonenveloped virus with a single-stranded circular DNA genome, which belongs to the Circoviridae family.PCV1 was first discovered in 1974 by Tisher as noncytopathic contaminant of the porcine kidney cell culture PK-15(ATCC-CCL33).Its complete genome was composed of 1759 nucleotides. However, PCV2 has been associated with postweaning multisystemic wasting syndrome(PMWS),which genome consists of 1767 or 1768 nucleotides. PMWS is an immunosupressive disease characterized by progressive weight loss and multisystemic pathological lesion in the 6-8 week-old piglets.PCV2 was first identified in China in 2000,and since then, the distribution of PMWS has spread in all over country, caused significant economic losses and present a threat to the pig industry. So there has significance on strengthening pathology detection and clinical diagnosis. This study included as following:The nucleic acid framents of PCV2 were detected from the tissues of clinical pigs with PMWS. The suspension was inoculated in PCV1 and PCV2 free PK-15 cells. After 8 passages, one isolate of porcine circovirus type 2 was isolated and named HN-PCV2.Purified virus was observed in transmission electron microscope. Virus-like particles were observed in the PK-15 cells infected PCV2,nothing in the PK-15 cells without infection. Specificity fluorescence in the PK-15 cells infected PCV2 was observed by IFA. Nothing in the PK-15 cells without infection.A pair of primers was designed, complete genome of the isolate was amplificated, and homology analysis of the nucleotides sequence and Phylogenetic tree compared with PCV1 and PCV2 in genbank. The result of sequence and analysis showed that HN-PCV2 belongs to Europe strains and the isolate's complete genome was composed of 1767 nucleotides, nucleotides containing 11 ORFs. Compared with PCV1 and PCV2 reference strains from Genbank, genome of HN-PCV2 isolate shared about 70% homology with PCV1, 94.0%~99.2% homology with PCV2. 96.1%~99.7% homology with PCV2 ORF1, 92.0%~99.3% homology with PCV2 ORF2, amino acid sequence were 95.2~98.1%,93.2%~99.1% respectively. According to genome sequences within ORF2 of porcine circovirus type 2 (PCV-2) published in Genbank, a pair of primers was designed, a SYBR-GreenⅠreal-time PCR which can detect porcine circovirus type 2 quickly and specially was established. The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration, melt curve was specificity and the correlation coefficient was 0.9988, the lowest copies which were detected were 1 copy perμL, and the quantitative PCR was more reproducibility and specificity than traditional PCR. The close-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 3 h or less.Heart, liver, spleen, lung .kidney and inguinal lymph nodes in the PMWS pigs were collected respectively, the PCV2 DNA in these organs were detected by traditional PCR, then detected quantitatively by the established real-time PCR. The results showed PCV2 existed widely inguinal lymph nodes, kidney and spleen, and the viral loads were higher than those of other organs, 7.39×1010 copies/g,1.80×1012 copies/g,8.43×109 copies/g respectively, the next in lung, the viral loads was 8.0×108 copies/g, the last in liver and heart, the test results were negative in the control pigs. The results also indicate that PCV2 mainly propagated in the immune organs, which led to lymphocyte depletion. The study provides significant value for clinical rapid diagnosis and the basis for the research of PCV2 pathopoiesis mechanism and tissue tropism.