Study On Amplification And Its Optimum Reaction Conditions Of E₂ Gene Of Hog Cholera Virus Guangxi Prevalence Strain

In this study, Hog Cholera Virus (HCV) specific primers Al/A2 was used to detect 58 clinical samples from some places in Guangxi with reverse transcrition-olymerase chain reaction (RT- CR). The results showed that 34 samples were positive and the positive rates were 58.62%. According to the published nucleotide sequence of genome of HCV, 4 pairs of specific primers, EP1/EP4, EP2/EP3, EP3/EPland EPS/EP6, were designed and synthesized. Primers EP1/EP4 and EP2/EP3 were matched, so the primers EP3/EP7 and EP5/EP6. The E2 gene fragment of HCV Guangxi prevalence strain was amplified in two parts with RT- CR method and the reaction conditions were optimized. The results demonstrated that Hog cholera virus lapinized Chinese strain (HCLV) and Shimen strain could be amplified with EP1/EP4 and EP2/EP3, but Guangxi prevalence strain not, while EP3/EP7 and EP5/EP6 could successfully amplify Guangxi prevalence strain, HCLV and Shimen strain. The reaction conditions could be optimized by under methods. lOul of the total RNA and 12. 5u1 of cDNA were used in reverse transcrition and PCR respectively, positive and negative primers were SOpmol respectively, EP3/EP7 and EP5/EP6 were denaturated at 570Cand 61 0C respectively with 35 cycles. Then both the product of PCR and its amplifing specificity amounted to the highest level. The successful amplification of E2 gene of Guangxi prevalence strain and the optimum reaction conditions play an important role in the further research of cloning and sequence analyzing on E2 gene.