Isolation And Identification Of Porcine Circovirus And Establishment Of Detection Methods

Porcine circovirus(PCV)is one of infectious disease sources in the world which was widesread focus on.There are two genotypes,PCV1 and PCV2. According to pathogenicity,antigenicity and nucleotide sequence,the porcine circovirus, which was gained from PK-15 cell line,was called PCV1,and the porcine circovirus,which was related with postweaning multisystemic wasting syndrome(PMWS),was called PCV2.In this study,a PCV2 strain was gained from swines,which was suspect to PMWS and from HeBei Baoding area.This strain was characterized by PCR and physicochemical affection,and a clinical diagnosis method was established,which was fast and precise.The PCV2 strain was getted from PK-15 cell line which was inoculated by lymphoglandulae inguinales, The organism was from swine which was suspect to PMWS.For cytopathic effect doesn't form in PK-15 cell line when porcine circovirus was inoculated,the PCV2 was harvested after inoculating for 72 hours and blind passaged for 4 generations.According to PCV2 gene order which were published in GenBank,specific primers were designed and synthesized,and ORF2 gene order of PCV2 was amplified. After DNA extracting and PCR amplification,a 485bp specific nucleinic acid fragment was getted.The TCID50 of PCV2 strain was determined by Immunoperoxidase Monolayer Assay(IPMA),physicochemical characteristics also be determined by this method.The result shows that the TCID50 of PCV2 strain has not remarkble changed after deal with 1 hour in PH3 or PH12,the strain has characteristics of strong resist acid and alkali,insensitivity for trypsinase,the difference of TCID50 are not reach to 2 after dealing with ether or chloroform.The result shows that PCV2 strain haven't peplos which was consistent with the bionomics of PCV2.Two pairs of specific primers were designed according to the sequences of PPV(VP2) and PRV(UL39),which were published in Genbank and Primer Premier 5.0 was used in the designe.PCV,PRV and PPV DNA could be amplified by the multipx PCR using these three sets of primers,yielding three specific fragments. By improving reactive condition of primer dosis,temperature and template dosis,better result getted ans specificity and reproducibility were fine.The PCR production of PCV2 was determined by XbaI,267bp and 228bp fragments were obtained,the pMD18T-PPV and pMD18T-PPV were determined by BamHI and HindIII, PPV 158 bp and PRV 219 bp fragments were getted.The sequencing result verifed the accuratissime of the fragments.In this study,double orientation latex agglutination text was established which was used to detect PCV2.The optimum condition of allergizing latex was definited by determining the density,temperature and time.The stability of the allergized latex was tested,which was contained preservative temperature and preservative time.The qualitative appreciation of latex agglutination antigen contains self-cohere detection ,which used PBS,BBS and sodium chloride,and specificity detection,which used PRV,swine plague positive serum,PCV negative serum and calf serum.It is revealed that the double orientation latex agglutination text is of high speciality and simplicity and makes the basis of serological diagnosis of PCV2.