Study On An Indirect ELISA For Detection Of Antibodies Against Classical Swine Fever Virus And On Real Time PCR For Classical Swine Fever Virus And Other 3 Viruses Which Could Induce Reproductive Troubles Of Pigs

The detection of CSFV antibody is the basis of establishing proper immune program and evaluating the effect of vaccination on swines. In this study, the four independent antigen regions of E2 gene were amplified from recombinant plasmid S21 by Polymerase Chain Reaction (PCR) and cloned into pMAL-p2X vector respectively. After identification, the positive recombinant plasmids were named as E2-abcd / pMAL-p2X, E2-ala2 / pMAL-p2X, E2-b / pMAL-p2X and E2-c / pMAL-p2X. Transfer these recombinant plasmids into 4 different hosts and then the E. coli BL21 — CodonPlus(DE3)-RP was proved to be the best. Soluble fusion proteins accounted for 15-30% of all proteins were obtained after the E. coli BL21-CodonPlus(DE3)-RP were induced by IPTG. These proteins were purified by affinity column and then were coated to the ELISA plates. The indirect ELISA to detect CSFV antibodies were developed in this study. The results showed that the assay has high sensitivity to CSFV positive serum comparing with negative serum.The detection of CSFV is essential to diagnose CSF. So conservative primers and probes were designed according to the conservative sequences of four viruses- CSFV, PRRSV, PRV and PPV which could induce reproductive troubles of pigs. Then two diplex real time PCR assays were developed to detect these 4 viruses. Among the 4 viruses, the assays had high specificity. To CSFV cDNA, when the sample was serial diluted, the detection limit was 2^-8 by real time PCR and 2^-9 by nest-PCR; to PRRSV cDNA, the detection limit was 2^-2 by real time PCR and 2" by nest-PCR; to PRV DNA, the detection limit was 10⁵, 10-fold times more sensitive than PCR and to PPV DNA the detection limit was 10^-6, 100-fold times more sensitive than PCR.