Detection And Application Study On Animal Coronavirus

The specific leader prime transcription mechanism of Coronavirus leads to high variation of antigen and high rate of recombination. It brings massive difficulties for diagnosis and control of Coronavirus. The establishment of effective detection method of animal Coronavirus is essential to control its prevalence .Referring to relative sequences of animal Coronavirus announced at GenBank , a universal PCR method was built up with a pair of universal prime which was designed according to conservative fragment of Coronavirus polymerase genes. The method of universal PCR established can detect the case of infection by Coronavirus and other group coronaviruses can also be detected and diagnosed quickly and briefly. The result showed that the method was specific and sensitive.449bp gene segments amplified by CCV and other coronaviruses was the same length with designation. and the sensitivity of experiment indicated that the method was as sensitive as normal PCR. The segment which was amplified by RNA polymerase genes was compared with relevant viruses. The result showed their homology was between 96% and 99%.moreover, the method of universal primer PCR was validity. Simultaneously, the segments amplified were compared with reference strain and analyzed by phylogenetic tree, their homology was between 56.69% and 99.55%.FCV,TGEV,FIPV,CCV,PRCV and HCV-229 attribute to an antigen group; BCV,HCV-Paris,and MHV-A59 to another;SARS is a new antigen group. All these were identical to articles reported.With the successful development of gene chip, different animals were innoculated with relevant Coronavirus. The cDNA of tissue samples were amplified by multiplex PCR and labeled by Cy3 , then their products were hybridized with relevant dot of gene chip . At the same time, samples were detected by RT-PCR and pathogenic isolation. The result showed that negative samples detected by latter two methods were positive outcome detected by gene chip; and that the method of RT-PCR was more sensitive than that of pathogenic isolation. Therefore, experiment confirmed that the detection of Coronavirus by gene chip was more sensitive and exact than other methods at gene chip application angle.According to the sequence of canine Coronavirus Tn449 strain announced at NCBI.890bp S gene fragment amplified by RT-PCR was compared with reference strains and analyzed. The result showed the homology of CCV Tn449 strain with UCD-1 strain and Emlo/02 strain was the lowest, In particular Emlo/02 strain's homology of nucleotide and amino acid with CCV Tn449 strain was 35.8% and 28.4% respectively. The homology of nucleotide and amino acid with other reference strains was 96.0%～87.3% and 96.6%～91.2% respectively. Simultaneously, The expression vector pET32a-CCV-S of S gene sequence was constructed to study the function of CCV S protein and diagnose CCV with recombined CCV S.