Establishment Of PCR Diagnosis Methods And Criterion For Porcine Circovirus

Porcine circovirus type2 (PCV2) was considered being associated with Postweaning Multisystemic Wasting Syndrome (PMWS), which was broken out all over the world in recent years.PMWS was clinically characterized by wasting and multisystemic pathologic damnification.furthermore,the infection of PCV2 can induced immunosuppression which can lead to the sequential inflection of other pathogens.The wide prevalence and spreading of the disease brought out serious threat to pig industry and huge loss in many nations and regions in the world. In the present study, This study established PCR method for porcine circovirus detection and two multiplex ploymerase chain reaction detection. was used for PCV2 detection DNA infected with PCV2 in henan province of China.1. A pair of primers was synthesized from the porcine circovirus DNA sequences published in GenBank. Through optimizing PCR conditions, the expected 487 bp fragment was amplified from DNA of PCV2-infected PK-15 cells, The amplified fragment was shown to be specific for PCV2 DNA after sequencing and digested by EcoRâ… .This method could detect the template DNA of 10fg at least. These results showed the PCR technique is a fast,simple,economical,specific and sentitive detection method.2. On the basis of single PCR for PCV2 and PRV established, by optimizing action conditions, The Multiplex PCR was developed.The results showed that two specific fragments of 488 bp for PCV2 and 355 bp for PRV could be amplified in the multiple PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PPV and control cultural cells(PK-15), This method could detect the template DNA of 100pg for PCV2 and 10pg for PRV, This method could differentiate PCV2, PRV and mixed infection.3.A multiplex ploymerase chain reaction was optimized to simultaneousely detecte three pathogens of PCV2,PPV and PRV Three sets of specific primers were designed according to the conservative sequences of PCV2, PPV and PRV. It was proved that the samples which contained PCV2,PPV and PRV could be amplified by the multiplex PCR using the three sets primers and it would yield three specific bands of 488bp(PCV2), 195bp(PPV) and 355bp(PRV).But no specific bands were amplified from other pathogenic viruses and control cultural cells(PK-15).As little as PCV2 50pg, PPV100pg and PRV10pg were detected using gelelectrophoresis in this multiplex PCR.This method could differentiate PCV2 infection, PPV infection, PRV infection.and mixed infection from the reproductive failure...