Diagnosis Establishment And Standard Formulation Of Polymerase Chain Reaction Detection For Porcine Parvovirus

Porcine parvovirus(PPV)causes reproductive failure in swine,manifested as embryonic resorption,fetal mummification,abortion and stillbirths.The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested.In this research,the PCR method and fluorescence quantitative PCR for the detection of PPV was developed.1.A pair of primers was synthesized from the porcine parvovirus DNA sequences published in GenBank.Through optimizing PCR conditions,the expected 322 bp fragment was amplified from DNA of PPV-infected PK-15 cells,The amplified fragment was shown to be specific for PPV DNA after sequencing and digested by BstXâ… .This method could detect the template DNA of 10fg at least(about the virus of 10^-4TCID₅₀).These results showed the PCR technique is a fast,simple,economical,specific and sentitive detection method.2.A pair of primers and the corresponding TaqMan probe were designed,the expected fragment was amplified from DNA of PPV-infected PK-15 cells,The purified PCR product was connected with pGEM-T Easy vector and then transformed into JM109.The standard recombinant plasmid was gained from positive bacterium clone.The plasmid PCR and plasmid sequencing showed that the expected fragment was successfully cloned.Series of diluted standard recombinant plasmid DNA specimen were amplified by real-time quantitative PCR,which indicated that there was a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen.Analysis of the dynamic curve,under this condition of the reaction,the sensitive degree is 90 copies(about the virus of 10^-6 TCID₅₀).The construction of real-time quantitative PCR provides the basis for the early and rapid detection and analysising the infected degree of PPV.3.On the basis of single PCR for PPV and PRV established,by optimizing action conditions,The Multiplex PCR was developed.The results showed that two specific fragments of 751 bp for PPV and 355 bp for PRV could be amplified in the multiple PCR simultaneously.DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells(PK-15),This method could detect the template DNA of 100pg(about the virus of 1 TCID₅₀)for PPV and 10pg(about the virus of 1 TCID₅₀)for PRV,This method could differentiate PPV,PRV and mixed infection.