Establishment Of PCR Assay For Detecting Porcine Circovirus Serotype 2 And Analysis On Genetic Variation Of PCV2 Gene From Gansu Isolate

Porcine circovirus type 2(PCV2) infection is an important infectious disease in pigs in recent years, which seriously hinders the development of pig industry. PCV2 infection can cause many diseases,such as Postweaning Multisystemic Wasting Syndrome (PMWS), Porcine Reproductive Disease Syndromes(PRDS), Porcine Dermatitis and Nephropathy Syndrome(PDNS), Congenital Tremors (CT), Interstitial Pneumonia(IP), Porcine Proliferative and Necrotic Pneumonia(PNP),and so on. PCV2 infection can depress the body immunity, cause secondary infection by other pathogenic microorganisms and aggravate the pathogenetic condition. The disease spread widely in many countries and areas and bring big economic loss in pig industry all over the world. There are a lot of reports about the prevalence of PCV2 in China in succession . And many borderline cases in clinic have been found in Gansu province. The report of Investigating serology has existed , whereas no having the proof of pathogeny diagnoses. In order to offer a credible mean to detect the pathogeny in lab,the method of PCR for PCV 2 has been established based on the rerults of our lab that can detect PCV2 fast. Meanwhile we analyse the PCV2 isolated from Gansu province on genetic variation as follows.1. Based on the full gene sequence of PCV2,a pair of specific primers (P1/P2) were designed and synthesized which can amplify the 262 bp specific fragments. In order to get the best result and hypersensitivity,the condition of amplification was regulated. The way of direct cross method has been used in order to make sure the best condition of amplification by gradient PCR to change the denaturation temperature and time, reannealing temperature and time, elongation time and circulation number. Finally, the PCR technique for detecting PCV2 has been established. The minimum content of DNA from specific fragments that obtained from the positive infected genome was 10-6ng/mL(about 100 PCV2 virus). The 75 samples(lung and ganglia lymphatica) of clinical invasion pigs that from Ganshu province was detected by this way ,and 39 samples were positive, and the positive rate was 52%.2. Based on the full gene sequence of PCV2,a pair of specific primers (P3/P4) were designed and synthesized. 6 positive samples were taken from the PVC2 positive samples in random. The amplification of the PCV2 full gene order was named Gansu1,Gansu2, Gansu3,Gansu4,Gansu5,Gansu6.Then the gene was cloned into pMD18-T vector. The sequencing result indicate that the lengh of genome Gansu5 is 1768 bp,all the other strains are 1767 bp. The sequence analysis software of DNAstar was used to make homology comparison and draw the phylogenetic trees of the 6 PCV2 full gene order and other PCV strains in Genbank .The results showed that the 6 isolated PCV2 strains shared 97.8%~99.4% nucleotide similarity; only shared 69.2%~70.3% nucleotide similarity with PCV1. ORF1 shared 97.6%~99.1% amino acids homogeneity and ORF2 shared 89%~99.2% compared with the other PCV2 strains in Genbank.