Establishment And Rudimentary Application Of Multi-PCR Of CAV-CPV

Two pairs of primers of a multi-PCR for CAV-CPV were designed with the aid of DNAsis software. By using the common primers within E3 and its flanking regions of CAV, CAV-1 could be amplified with a fragment of 513bp DNA in length, while CAV-2 gave a 1023bp DNA fragment; CPV could be amplified with a 719bp fragment by a pair of primers specific for canine parvovirus NS1 region in the same reaction tube. The specificity and their identity to other CAV and CPV isolates of the PCR products were confirmed by the size of the DNA fragments on gel electrophoresis and the DNA restriction digestion. Simultaneously no specific fragment was amplified from MDCK, F81 cells, rabbies virus and pseudorabies virus cultures. Thirty clinical samples were detected at the same time by electron microscopy, HA/HI, virus isolation and multi-PCR. The results of the PCR were in agreement with that of virus isolation, and more sensitive than the other two methods.One pair of PCR primers was designed to discriminate the virulent strains of CAV-l.The conserved regions E3 of the virulent and the attenuated CAV-1 are different. After amplificating, a 569bp fragment was obtained from the virulent strain while 244bp fragment from the attenuated strain. The products of PCR were examined by electrophoresis, restriction enzyme digestion and sequencing assay. The results showed that the length of the fragments, enzyme sites and sequence were as the same as those of virulent strain and attenuated strain of CAV-1. No specific fragment was amplified from MDCK control cells and canine adenovirus 2 culture. The PCR of CAV-1 was proved to be sensitive and specific. It is possible that this method may be used in discriminating the virulent/attenuated strains of the CAV-1 and in clinical diagnosis of CAV-1 infection.In order to remove the substances inhibiting the PCR in the fecal specimens, we developed a method to purify virus in the feces. Staphylococcal protein A can bind to the Fc fragment of canine IgG, so sensitized staphylococcus aureus with anti-serum against CAV /CPV can bind CAV and CPV. The conditions of the sensitization and the incubated time of sensitized staphylococcus aureus with35virus were optimized. 8 known positive samples which is negative by directing PCR were purified under the selected conditions and examined by the multi-PCR, the results are all positive, implicating the method is effective, and can be used with the multi-PCR in clinical diagnosis.The method described here is very practicable and sensitive, specific, simple and cheap. It would be used in diagnosis of CAV-1, CAV-2 and CPV.