Development And Application Of A Multiplex PCR For Detecting Porcine Parvovirus, Pseudorabies Virus And Porcine Circovirus Type 2

Porcine parvovirus , porcine pseudorabies and porcine circovirus are three main threat to the development of swine-breeding industry.Beacause sigle or mixed infection of the three virus both can cause diseases which can give rise to reproductive failure or(and) immunosuppression, it provides a convenient chance for secondary infection by other pathogens and enven causes epidemic diseases in pigs wich can lead to tremendous economic losses ,and may arise public health problems. PCR with rapid,specific and sensitive advantages exhibits obvious preponderance in specificit detection of infectious agents in animals since it was invent.And it is applied widely in animal disease diagnosis and pathogen detection which has made important contributions to disease prevention and control. At present,the cases of mixed infection of pathogen are increasing,and common分子molecular detection of pathogen has show its own shortage of time consuming ,high cost and labour power when we need detect one specimen repeatedly.Mutiplex PCR not only preserve advantages of routine PCR,but also owns advantages of convenience shortcut and sensitiveness which meet the need of fast diagonosis for multiple diseases in production.We found that sigle or mixed infection of PPV,PRV and PCV-2 is common in the course for the survey of viral pathogen in some breeding enterprises in this research.So we plan to establish the mutiplex PCR for detection of PPV,PRV and PCV-2,in purpose for providing fast detection of the three pathogens an available method .(1)Specific primers for DNA viruses, namely, porcine parvovirus (PPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV-2),were synthesized according to the published virus conserved gene sequence of PPV, PRV and PCV-2 in GenBank and used by DNAStar and Oligo 6.0 software . We establish a mutiplex PCR method for the detection of PPV, PRV and PCV-2 by optimizing concentration of primers,annealing temperature and component of PCR.Sensibility test shows that the susceptibility of PRV and PCV-2 reach the level of pg.With PCV-1, bovine viral diarrhea virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, Escherichia coli, PPV, PRV, PCV-2 as a template for multiple PCR were detected,the results show that the only PPV, PRV and PCV-2 virus in a single or multiplex PCR were amplified by their specific band.(2)The blood samples collected from 286 pig farms in Shaanxi Province were viral detection by this method. Clinically healthy pigs from a blood sample PPV, PRV and PCV-2 test results indict that PCV-2 in normal pigs have a certain percentage of presence, meanwhile several farms also exist the phenomenon of the PRV and PCV-2 dual infection.Swine from the clinical illness were detected compound, It was serous of PRV+PCV-2 combined infection in diseased pigs,and PPV+PRV+PCV-2 combined infection also were detected in diseased pigs. The coincidence rate of multiplex PCR test and single PCR test is more than 99%, illutstrating that The developed novel multiplex PCR will be a tool used for the detection of clinical samples for diagnosis reference.