Detection Of PRV,PPV And PCV2 Infection Using PCR

1. 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 1030bp(PCV2) 582bp(PPV) and 372bp(PRV).But no specific bands were amplified from other pathogenic viruses and bacterium.As little as PCV2 0.1pg PPV 10-3.5 TCID50 and PRV 10-4.6TCID50 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.2. A multiplex ploymerase china reaction diagnostic kit was optimized to simultaneousely detected Vaccine from Wild-type isolates of PrV.Three sets of specific primers were designed according to the conservative sequences of PRV.Using these three sets primers, three specific bands of TK(277bp)> gE (144bp) and gB(372bp) could be yielded.But no specific bands were amplified from other pathogenic viruses and bacterium.lt was proved that the samples which contained Vaccine and Wild-type of PRV could be amplified by the PCR Kit.As little as TK 10-4.6 TCID50 gE10-3.6 TCID50 and gB 10-4.6 TCID50 were detected using this PCR Kit. And we optimized the PCR Kit. It kept effective stored at -20 C for 4 months.The PCR Kit will be useful for the rapid detection of PrV in clinical laboratories and for the Eradication of Pseudorabies Virus.3. From January of 2002,an epidemiological investigation on infection of Pcv2 was carried out in 17 intensive swine farms in Anhui, Jangsu, Jiangxi, Guangdong, Zhejiang, Shandong and Hubei provence. This investigation includes the normal swine farms andthe infectious swine farms of PMWS.PCV2 in tissues was detected by PCR. It was shown that the infection of PCV2 was 24.6% in the normal farms and 60.6% in the infectious swine farms.It proved that the infection of PCV2 existed widely in swine farms in China.4. According to the published genomic sequences of PCV2, two pairs of PCV2 specific primers were designed, and three PCV2 complete genomes were amplified by polymerase chain reaction (PCR).Three tissue samples postive for PCV2 tested by PCR were inoculated into PK-15 cells, respectively. After six blind passsages, the propagation of PCV2 was identified by PCR, respectively. The results showed that PCV2 specific DNAs (1030bp) were detected by PCR in all the three inoculated PK-15 cells. The three PCV2 isolates identified in the present study were designated PCV-SHO PCV-JS and PCV-SHM respectively according to the different isolated regions. Three recombinant plasmids containing the corresponding complete genome of PCV2 isolates were obtained. The sequences of cloned PCR products were analysed, and the complete genomes of the three PCV2 isolates were determined, all of them are 1767bp in length. The three sequences were compared with other PCV isolates in the Genbank, the results indicated that three PCV2 isolates sequenced in the present study are closely related to other PCV2 isolates in the Genbank, displaying 94.5%~100% nucleotide sequence identities.Although the genome of PCV2 isolates is generally stable among different isolates, PCV2 isolates from different geographic regions vary in the genomic sequences.5. Pig IgG, which was purified from pig sera by water dilution, (NH4)2SO4 precipitation, alcohol precipitation and column chromato graphy with sephadexG-200,was used as antigen to immune BALB/C mice.By fusion of SP2/0 myelom a cells and spleen cells of these mice,two monoclonal antibodies against pig IgG in indirect ELISA were screened and designated as 1 A10B4 and 3B11E2.Both of them reacted with an approximately 60KD protein in Westem-blotting,indicating that they recognized the heavy chains of pig IgG.Inindirect ELISA,the titers of 1A10B4 and 3B11E2 ascitic fluids were 1:2000 and 1:400...